Fusion proteins for treating pathological blood clots

ABSTRACT

The present disclosure provides various molecular constructs having a targeting element and an effector element. Methods for treating various diseases using such molecular constructs are also disclosed.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present disclosure relates to the field of pharmaceuticals; more particularly, to multi-functional molecular constructs, e.g., those having targeting and effector elements for delivering the effector (e.g., therapeutic drug) to targeted sites.

2. Description of the Related Art

The continual advancement of a broad array of methodologies for screening and selecting monoclonal antibodies (mAbs) for targeted antigens has helped the development of a good number of therapeutic antibodies for many diseases that were regarded as untreatable just a few years ago. According to Therapeutic Antibody Database, approximately 2,800 antibodies have been studied or are being planned for studies in human clinical trials, and approximately 80 antibodies have been approved by governmental drug regulatory agencies for clinical uses. The large amount of data on the therapeutic effects of antibodies has provided information concerning the pharmacological mechanisms how antibodies act as therapeutics.

One major pharmacologic mechanism for antibodies acting as therapeutics is that, antibodies can neutralize or trap disease-causing mediators, which may be cytokines or immune components present in the blood circulation, interstitial space, or in the lymph nodes. The neutralizing activity inhibits the interaction of the disease-causing mediators with their receptors. It should be noted that fusion proteins of the soluble receptors or the extracellular portions of receptors of cytokines and the Fc portion of IgG, which act by neutralizing the cytokines or immune factors in a similar fashion as neutralizing antibodies, have also been developed as therapeutic agents.

Several therapeutic antibodies that have been approved for clinical applications or subjected to clinical developments mediate their pharmacologic effects by binding to receptors, thereby blocking the interaction of the receptors with their ligands. For those antibody drugs, Fc-mediated mechanisms, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytolysis (CMC), are not the intended mechanisms for the antibodies.

Some therapeutic antibodies bind to certain surface antigens on target cells and render Fc-mediated functions and other mechanisms on the target cells. The most important Fc-mediated mechanisms are antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytolysis (CMC), which both will cause the lysis of the antibody-bound target cells. Some antibodies binding to certain cell surface antigens can induce apoptosis of the bound target cells.

The concept and methodology for preparing antibodies with dual specificities germinated more than three decades ago. In recent year, the advancement in recombinant antibody engineering methodologies and the drive to develop improved medicine has stimulated the development bi-specific antibodies adopting a large variety of structural configurations.

For example, the bi-valent or multivalent antibodies may contain two or more antigen-binding sites. A number of methods have been reported for preparing multivalent antibodies by covalently linking three or four Fab fragments via a connecting structure. For example, antibodies have been engineered to express tandem three or four Fab repeats.

Several methods for producing multivalent antibodies by employing synthetic crosslinkers to associate, chemically, different antibodies or binding fragments have been disclosed. One approach involves chemically cross-linking three, four, and more separately Fab fragments using different linkers. Another method to produce a construct with multiple Fabs that are assembled to one-dimensional DNA scaffold was provided. Those various multivalent Ab constructs designed for binding to target molecules differ among one another in size, half-lives, flexibility in conformation, and ability to modulate the immune system. In view of the foregoing, several reports have been made for preparing molecular constructs with a fixed number of effector elements or with two or more different kinds of functional elements (e.g., at least one targeting element and at least one effector element). However, it is often difficult to build a molecular construct with a particular combination of the targeting and effector elements either using chemical synthesis or recombinant technology. Accordingly, there exists a need in the related art to provide novel molecular platforms to build a more versatile molecule suitable for covering applications in a wide range of diseases.

SUMMARY

<I > Peptide Core-Based Multi-Arm Linkers

In the first aspect, the present disclosure is directed to a linker unit that has at least two different functional elements linked thereto. For example, the linker unit may have linked thereto two different effector elements, one targeting element and one effector element, or one effector element and a polyethylene glycol (PEG) chain for prolonging the circulation time of the linker unit. The present linker unit is designed to have at least two different functional groups such that the functional elements can be linked thereto by reacting with the respective functional groups. Accordingly, the present linker unit can serve as a platform for preparing a molecular construct with two or more functional elements.

According to various embodiments of the present disclosure, the linker unit comprises a center core and a plurality of linking arms. The center core is a polypeptide core comprising (1) a plurality of lysine (K) resides, in which each K residue and a next K residue are separated by a filler sequence comprising glycine (G) and serine (S) residues, and the number of K residues ranges from 2 to 15; or (2) the sequence of (X_(aa)−K)_(n), where X_(aa) is a PEGylated amino acid having 2 to 12 repeats of ethylene glycol (EG) unit, and n is an integral from 2 to 15. Optionally, the filler sequence consists of 2 to 20 amino acid residues. In various embodiments, the filler sequence may have the sequence of GS, GGS, GSG, or SEQ ID NOs: 1-16. According to some embodiments of the present disclosure, the center core comprises 2-15 units of the sequence of G₁₋₅SK; preferably, the center core comprises the sequence of (GSK)₂₋₁₅. Each of the linking arms is linked to the K residues of the center core via forming an amide linkage between the K residue and the linking arm. The linking arm linked to the center core has a maleimide, an N-hydroxysuccinimidyl (NHS) group, an azide group, an alkyne group, a tetrazine group, a cyclooctene group, or a cyclooctyne group at its free-terminus. Also, the amino acid residue at the N- or C-terminus of the center core has an azide group or an alkyne group; alternatively or additionally, the amino acid residue at the N- or C-terminus of the center core is a cysteine (C) residue, in which the thiol group of the amino acid residue is linked with a coupling arm having an azide group, an alkyne group, a tetrazine group, a cyclooctene group, or a cyclooctyne group at the free terminus of the coupling arm.

According to various embodiments of the present disclosure, the linker unit further comprises a plurality of first elements. In some embodiments, each of the first elements is linked to one of the linking arms via forming an amide bound between the linking arm and the first element. In other embodiments, each of the first elements is linked to one of the linking arms via thiol-maleimide reaction, copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction, strained-promoted azide-alkyne click chemistry (SPAAC) reaction, or inverse electron demand Diels-Alder (iEDDA) reaction occurred between the linking arm and the first element.

According to some embodiments of the present disclosure, when the plurality of first elements are respectively linked to the plurality of linking arms via CuAAC or SPAAC reaction, then the amino acid residue at the N- or C-terminus of the center core is a cysteine residue, and the free terminus of the coupling arm is the tetrazine or the cyclooctene group. According to other embodiments of the present disclosure, when the plurality of first elements are respectively linked to the plurality of linking arms via iEDDA reaction, then the amino acid residue at the N- or C-terminus of the center core has the azide or the alkyne group, or the amino acid residue at the N- or C-terminus of the center core is a cysteine residue, and the free terminus of the coupling arm is the azide, the alkyne, or the cyclooctyne group.

In some embodiments, the linking arm is a PEG chain, preferably having 2 to 20 repeats of EG units. In other embodiments, the coupling linking arm is a PEG chain, preferably having 2 to 12 repeats of EG units.

Regarding amino acid residues having the azide group, non-limiting examples of said amino acid residues include L-azidohomoalanine (AHA), 4-azido-L-phenylalanine, 4-azido-D-phenylalanine, 3-azido-L-alanine, 3-azido-D-alanine, 4-azido-L-homoalanine, 4-azido-D-homoalanine, 5-azido-L-ornithine, 5-azido-d-ornithine, 6-azido-L-lysine, and 6-azido-D-lysine. As to the amino acid residues having the alkyne group, illustrative examples thereof include L-homopropargylglycine (L-HPG), D-homopropargylglycine (D-HPG), and beta-homopropargylglycine (β-HPG).

When the amino acid residues at the N- or C-terminus of the center core is the cysteine residue, the cyclooctene group at the free terminus of the coupling arm may be, a trans-cyclooctene (TCO) group, while the cyclooctyne group at the free terminus of the coupling arm may be a dibenzocyclooctyne (DBCO), difluorinated cyclooctyne (DIFO), bicyclononyne (BCN), or dibenzocyclooctyne (DICO) group. Alternatively, the tetrazine group at the free terminus of the coupling arm includes, but is not limited to, 1,2,3,4-tetrazine, 1,2,3,5-tetrazine, and 1,2,4,5-tetrazine, and derivatives thereof, such as, 6-methyl tetrazine.

According to various optional embodiments of the present disclosure, the first element is an effector element suitable for eliciting an intended effect (e.g., a therapeutic effect) in a subject. Alternatively, the first element may be a targeting element for directing the linker unit to the site of interest. According to the embodiments of the present disclosure, the first element is a single-chain variable fragment (scFv) specific for fibrin.

Optionally, the linker unit further comprises a second element that is different from the first elements. In some embodiments, the second element has an azide or alkyne group, so that it is linked to the center core or the coupling arm by coupling with the corresponding alkyne or azide group of the center core or the coupling arm via CuAAC reaction. Alternatively, in some embodiments, the second element having an azide or cyclooctyne group is linked to the center core or the coupling arm by coupling with the corresponding cyclooctyne or azide group of the center core or the coupling arm via SPAAC reaction. Still alternatively, in certain embodiments, the second element having a tetrazine or cyclooctene group is linked to the center core or the coupling arm by coupling with the corresponding cyclooctene or tetrazine group of the center core or the coupling arm via iEDDA reaction. According to the embodiments of the present disclosure, the second element is a tissue plasminogen activator or an inhibitor of Factor Xa or thrombin. Non-limiting examples of the tissue plasminogen activators include, alteplase, reteplase, tenecteplase, and lanoteplase. The inhibitor of Factor Xa is selected from the group consisting of, apixaban, edoxaban, and rivaroxaban. The inhibitor of thrombin can be argatroban or melagatran.

In certain embodiments, the linker unit further comprises an optional third element that is different from the first and second elements. In the case where the second element is directly linked to the center core, the other terminus (i.e., the free terminus that is not linked with the second element) of the center core is optionally a cysteine residue, which can be used to introduce an optional third element. Specifically, the thiol group of the cysteine residue is reacted with a maleimide group of a PEG chain; and the thus-linked PEG chain is designated as the coupling arm, which has a tetrazine group or a cyclooctene group at its free terminus. Accordingly, the third element is then linked to the coupling arm via iEDDA reaction. Preferably, the third element is an element for improving the pharmacokinetic property of the linker unit. One example of the element for improving the pharmacokinetic property is a long PEG chain having a molecular weight of about 20,000 to 50,000 Daltons.

<II> Uses of Peptide Core-Based Multi-Arm Linkers

The linker unit according to the first aspect of the present disclosure may find its utility in clinical medicine for the treatment of various diseases. Hence, the second aspect of the present disclosure is directed to a method for treating these diseases. According to various embodiments of the present disclosure, the method for treating a particular disease includes the step of administering to the subject in need thereof a therapeutically effective amount of the linker unit according to the above-mentioned aspect and embodiments of the present disclosure. As could be appreciated, said linker unit may be administered in a pharmaceutical formulation, which comprises a pharmaceutically-acceptable excipient suitable for the intended or desired administration route, in addition to the present linker unit.

According to some embodiments of the present disclosure, the present linker unit is useful in preventing the formation of blood clot. In these embodiments, the first element is an scFv specific for fibrin, and the second element is the inhibitor of Factor Xa or thrombin. The inhibitor of Factor Xa is selected from the group consisting of, apixaban, edoxaban, and rivaroxaban. The inhibitor of thrombin can be argatroban or melagatran.

According to other embodiments of the present disclosure, the linker units suitable for treating thrombosis comprise an scFv specific for fibrin as the first element, and a tissue plasminogen activator as the second element. Non-limiting examples of the tissue plasminogen activators include, alteplase, reteplase, tenecteplase, and lanoteplase.

<III> Fc-Based Molecular Construct for Preventing the Formation of Blood Clot and Treating Thrombosis and Uses Thereof

In the fifth aspect, the present disclosure is directed to a fragment crystallizable (Fc)-based molecular construct that has at least one targeting element and at least one effector element linked, directly or indirectly, to a CH2-CH3 domain of an immunoglobulin. Targeting and effector elements of the present Fc-based molecular constructs are specifically selected such that these Fc-based molecular constructs are suitable for use in the formation of blood clots and the treatment of thrombosis, or for use in the manufacture of a medicament for such uses. As could be appreciated, methods for preventing the formation of blood clots and for treating thrombosis using such Fc-based molecular constructs also fall within the aspect of the present disclosure.

According to certain embodiments of the present disclosure, the Fc-based molecular construct comprises a pair of CH2-CH3 segments of an IgG.Fc, a pair of effector elements, and a pair of targeting elements. The pair of effector element is a tissue plasminogen activator or a drug bundle comprising a plurality of molecules of an inhibitor of Factor Xa or an inhibitor of thrombin, while the pair targeting elements is an antibody fragment specific for fibrin.

In the case where the effector element is the tissue plasminogen activator (e.g., alteplase, reteplase, tenecteplase, and lanoteplase), then the pair of effector elements is linked to the N-termini of the pair of CH2-CH3 segments, and the pair of targeting elements is linked to the C-termini of the pair of CH2-CH3 segments, or vice versa. Alternatively, when the effector element is a drug bundle, then the pair of effector elements is linked to the C-termini of the pair of CH2-CH3 segments, and the pair of targeting elements is linked to the N-termini of the pair of CH2-CH3 segments.

In certain embodiments, the pair of CH2-CH3 segments is derived from human IgG heavy chain γ4 or human IgG heavy chain γ1.

In some examples, the pair of the targeting elements takes a Fab configuration (i.e., consisting of the V_(H)—CH1 domain and the V_(L)—Cκ domain); this Fab fragment is linked to the N-termini of the first and second heavy chains, so that the Fc-based molecular construct adopts an IgG configuration. In these cases, the pair of effector elements is linked to the C-termini of the pair of CH2-CH3 segments.

According to some optional embodiments, the effector elements are drug bundles based on linker units. Such drug bundles are advantageous at least in that they can be manufactured separately before being conjugated to the antibody molecules, thus avoiding subjecting drug molecules under harsh chemical conditions for the direct conjugation with the antibody molecules. According to various embodiments of the present disclosure, the drug bundle comprises a plurality of inhibitors associated with blood clotting, such as Factor Xa inhibitors (e.g., apixaban, edoxaban, and rivaroxaban) and thrombin inhibitors (e.g., argatroban and melagatran). As an example, rather than a limitation, these Fc-based molecular constructs are useful in the prevention of blood clotting.

According to certain embodiments, the present Fc-based molecular construct further comprises a peptide extension and a coupling arm. Specifically, the peptide extension has the sequence of (G₂₋₄S)₂₋₈C and is linked to the C-terminus of one of the pair of CH2-CH3 segments. In such cases, the coupling arm is linked to the C-terminus of the peptide extension via thiol-maleimide reaction occurred therebetween. Also, before being conjugated with the drug bundle, the free terminus of the coupling arm (that is, the terminus that is not linked to the cysteine residue) is modified with an alkyne, azide, strained alkyne, or tetrazine group, so that the drug bundle is linked thereto via inverse electron demand Diels-Alder (iEDDA) reaction or the strain-promoted azide-alkyne click chemistry (SPAAC) reaction or Copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction occurred therebetween.

According to some optional embodiments, the drug bundle comprises a center core and a plurality of linking arms. The center core may be a compound having a plurality of amine groups or a polypeptide comprising a plurality of lysine (K) residues, according to various embodiments of the present disclosure. Each of the linking arms has one terminus that is linked to the center core by reacting with the amine groups of the compound core or the K residues of the polypeptide core. The linking arm also carries a maleimide group at the free terminus thereof, wherein each of the molecules (e.g., molecules of inhibitors associated with blood clotting) is linked to the center core through the linking arm by reacting with the maleimide group.

In the case where the center core is the polypeptide core, then the amino acid residue at the N- or C-terminus of the center core is a cysteine residue or has an azide group or an alkyne group.

For polypeptide cores with a terminal amino acid residue having the azide group or the alkyne group, the drug bundle may be linked to the peptide extension via the CuAAC reaction occurred between said terminal residue and the C-terminus of the peptide extension.

Methods for preventing blood clot formation and/or treating thrombosis in a subject in need thereof comprise the step of administering to the subject an effective amount of the molecular construct of this aspect.

BRIEF DESCRIPTION OF THE DRAWINGS

The present description will be better understood from the following detailed description read in light of the accompanying drawings briefly discussed below.

FIG. 1A to FIG. 1K are schematic diagrams illustrating linker units according to certain embodiments of the present disclosure.

FIG. 2 is a schematic diagram illustrating a linker unit having a compound core.

FIGS. 3A to 3C are schematic diagrams illustrating Fc-based molecular constructs according to various embodiments of the present disclosure.

FIGS. 4A and 4B are schematic diagrams illustrating Fc-based molecular constructs according to various embodiments of the present disclosure.

FIG. 5 shows the mass spectrometry MALDI-TOF result of a peptide core-based linker-unit carrying one linking arm with TCO group and five PEG6 linking arms with maleimide groups, according to one working example of the present disclosure.

FIG. 6 shows the mass spectrometry MALDI-TOF result of the apixaban-PEG₃-S—S-PEG₃-apixaban synthetized according to one working example of the present disclosure.

FIG. 7 shows the mass spectrometry MALDI-TOF result of the argatroban-PEG₃-S—S-PEG₃-argatroban synthetized according to one working example of the present disclosure.

FIGS. 8A, 8B, and 8C respectively show the results of SDS-PAGE, MALDI-TOF and ELISA analysis of purified 102-10 scFv specific for human fibrin, according to one working example of the present disclosure.

FIG. 9A and FIG. 9B respectively show the results of phage titer analysis and single colony ELISA analysis of phage-displayed scFvs specific for human fibrin, according to one working example of the present disclosure.

FIG. 10A and FIG. 10B respectively show the results of SDS-PAGE and MALDI-TOF analysis of purified reteplase, according to one working example of the present disclosure.

FIG. 11 shows the result of mass spectrometric analysis of TCO-conjugated reteplase, according to one working example of the present disclosure.

FIG. 12 shows the result of SDS-PAGE analysis of a targeting linker-unit with one free tetrazine functional group and a set of three scFvs specific for human fibrin, according to one working example of the present disclosure.

FIG. 13 shows the MS result on a single linker-unit molecular construct with three scFvs specific for human fibrin (as targeting elements) and one reteplase molecule (as the effector element), according to one working example of the present disclosure.

FIG. 14 shows the result of inhibition assay of apixaban-PEG₃-SH molecule, according to one working example of the present disclosure.

FIG. 15 shows the result of SDS-PAGE analysis of purified recombinant 2-chain (reteplase)-hIgG4.Fc fusion protein, according to one working example of the present disclosure.

FIG. 16 shows the result of SDS-PAGE analysis of purified recombinant 2-chain (reteplase)-hIgG4.Fc-(scFv α fibrin) fusion protein, according to one working example of the present disclosure.

FIG. 17 shows the result of SDS-PAGE analysis of purified recombinant 2-chain (reteplase)-(scFv α fibrin) fusion protein, according to one working example of the present disclosure.

FIG. 18 shows the result of SDS-PAGE analysis of purified recombinant 2-chain (TNK-tPA)-IgG4.Fc fusion protein, according to one working example of the present disclosure.

FIG. 19 shows the assay results of protease activity of recombinant 2-chain (reteplase)-hIgG4.Fc, 2-chain (reteplase)-hIgG4.Fc-(scFv α fibrin) and (reteplase)-(scFv a fibrin), according to one working example of the present disclosure.

In accordance with common practice, the various described features/elements are not drawn to scale but instead are drawn to best illustrate specific features/elements relevant to the present invention. Also, like reference numerals and designations in the various drawings are used to indicate like elements/parts, where possible.

DETAILED DESCRIPTION OF THE INVENTION

The detailed description provided below in connection with the appended drawings is intended as a description of the present examples and is not intended to represent the only forms in which the present example may be constructed or utilized. The description sets forth the functions of the example and the sequence of steps for constructing and operating the example. However, the same or equivalent functions and sequences may be accomplished by different examples.

For convenience, certain terms employed in the specification, examples and appended claims are collected here. Unless otherwise defined herein, scientific and technical terminologies employed in the present disclosure shall have the meanings that are commonly understood and used by one of ordinary skill in the art.

Unless otherwise required by context, it will be understood that singular terms shall include plural forms of the same and plural terms shall include the singular. Specifically, as used herein and in the claims, the singular forms “a” and “an” include the plural reference unless the context clearly indicated otherwise. Also, as used herein and in the claims, the terms “at least one” and “one or more” have the same meaning and include one, two, three, or more. Furthermore, the phrases “at least one of A, B, and C”, “at least one of A, B, or C” and “at least one of A, B and/or C,” as use throughout this specification and the appended claims, are intended to cover A alone, B alone, C alone, A and B together, B and C together, A and C together, as well as A, B, and C together.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in the respective testing measurements. Also, as used herein, the term “about” generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term “about” means within an acceptable standard error of the mean when considered by one of ordinary skill in the art. Other than in the operating/working examples, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages such as those for quantities of materials, durations of times, temperatures, operating conditions, ratios of amounts, and the likes thereof disclosed herein should be understood as modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and attached claims are approximations that can vary as desired. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Ranges can be expressed herein as from one endpoint to another endpoint or between two endpoints. All ranges disclosed herein are inclusive of the endpoints, unless specified otherwise.

This present disclosure pertains generally to molecular constructs, in which each molecular construct comprises a targeting element (T) and an effector element (E), and these molecular constructs are sometimes referred to as “T-E molecules”, “T-E pharmaceuticals” or “T-E drugs” in this document.

As used herein, the term “targeting element” refers to the portion of a molecular construct that directly or indirectly binds to a target of interest (e.g., a receptor on a cell surface or a protein in a tissue) thereby facilitates the transportation of the present molecular construct into the interested target. In some example, the targeting element may direct the molecular construct to the proximity of the target cell. In other cases, the targeting element specifically binds to a molecule present on the target cell surface or to a second molecule that specifically binds a molecule present on the cell surface. In some cases, the targeting element may be internalized along with the present molecular construct once it is bound to the interested target, hence is relocated into the cytosol of the target cell. A targeting element may be an antibody or a ligand for a cell surface receptor, or it may be a molecule that binds such antibody or ligand, thereby indirectly targeting the present molecular construct to the target site (e.g., the surface of the cell of choice). The localization of the effector (therapeutic agent) in the diseased site will be enhanced or favored with the present molecular constructs as compared to the therapeutic without a targeting function. The localization is a matter of degree or relative proportion; it is not meant for absolute or total localization of the effector to the diseased site.

According to the present invention, the term “effector element” refers to the portion of a molecular construct that elicits a biological activity (e.g., inducing immune responses, exerting cytotoxic effects and the like) or other functional activity (e.g., recruiting other hapten tagged therapeutic molecules), once the molecular construct is directed to its target site. The “effect” can be therapeutic or diagnostic. The effector elements encompass those that bind to cells and/or extracellular immunoregulatory factors. The effector element comprises agents such as proteins, nucleic acids, lipids, carbohydrates, glycopeptides, drug moieties (both small molecule drug and biologics), compounds, elements, and isotopes, and fragments thereof.

Although the terms, first, second, third, etc., may be used herein to describe various elements, components, regions, and/or sections, these elements (as well as components, regions, and/or sections) are not to be limited by these terms. Also, the use of such ordinal numbers does not imply a sequence or order unless clearly indicated by the context. Rather, these terms are simply used to distinguish one element from another. Thus, a first element, discussed below, could be termed a second element without departing from the teachings of the exemplary embodiments.

Here, the terms “link,” “couple,” and “conjugates” are used interchangeably to refer to any means of connecting two components either via direct linkage or via indirect linkage between two components.

The term “polypeptide” as used herein refers to a polymer having at least two amino acid residues. Typically, the polypeptide comprises amino acid residues ranging in length from 2 to about 200 residues; preferably, 2 to 50 residues. Where an amino acid sequence is provided herein, L-, D-, or beta amino acid versions of the sequence are also contemplated. Polypeptides also include amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. In addition, the term applies to amino acids joined by a peptide linkage or by other, “modified linkages,” e.g., where the peptide bond is replaced by an α-ester, a β-ester, a thioamide, phosphoramide, carbomate, hydroxylate, and the like.

In certain embodiments, conservative substitutions of the amino acids comprising any of the sequences described herein are contemplated. In various embodiments, one, two, three, four, or five different residues are substituted. The term “conservative substitution” is used to reflect amino acid substitutions that do not substantially alter the activity (e.g., biological or functional activity and/or specificity) of the molecule. Typically, conservative amino acid substitutions involve substitution one amino acid for another amino acid with similar chemical properties (e.g., charge or hydrophobicity). Certain conservative substitutions include “analog substitutions” where a standard amino acid is replaced by a non-standard (e.g., rare, synthetic, etc.) amino acid differing minimally from the parental residue. Amino acid analogs are considered to be derived synthetically from the standard amino acids without sufficient change to the structure of the parent, are isomers, or are metabolite precursors.

In certain embodiments, polypeptides comprising at least 80%, preferably at least 85% or 90%, and more preferably at least 95% or 98% sequence identity with any of the sequences described herein are also contemplated.

“Percentage (%) amino acid sequence identity” with respect to the polypeptide sequences identified herein is defined as the percentage of polypeptide residues in a candidate sequence that are identical with the amino acid residues in the specific polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percentage sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, sequence comparison between two polypeptide sequences was carried out by computer program Blastp (protein-protein BLAST) provided online by Nation Center for Biotechnology Information (NCBI). The percentage amino acid sequence identity of a given polypeptide sequence A to a given polypeptide sequence B (which can alternatively be phrased as a given polypeptide sequence A that has a certain % amino acid sequence identity to a given polypeptide sequence B) is calculated by the formula as follows:

$\frac{X}{Y} \times 100\%$

where X is the number of amino acid residues scored as identical matches by the sequence alignment program BLAST in that program's alignment of A and B, and where Y is the total number of amino acid residues in A or B, whichever is shorter.

The term “PEGylated amino acid” as used herein refers to a polyethylene glycol (PEG) chain with one amino group and one carboxyl group. Generally, the PEGylated amino acid has the formula of NH₂—(CH₂CH₂O)_(n)—COOH. In the present disclosure, the value of n ranges from 1 to 20; preferably, ranging from 2 to 12.

As used herein, the term “terminus” with respect to a polypeptide refers to an amino acid residue at the N- or C- end of the polypeptide. With regard to a polymer, the term “terminus” refers to a constitutional unit of the polymer (e.g., the polyethylene glycol of the present disclosure) that is positioned at the end of the polymeric backbone. In the present specification and claims, the term “free terminus” is used to mean the terminal amino acid residue or constitutional unit is not chemically bound to any other molecular.

The term “antigen” or “Ag” as used herein is defined as a molecule that elicits an immune response. This immune response may involve a secretory, humoral, and/or cellular antigen-specific response. In the present disclosure, the term “antigen” can be any of a protein, a polypeptide (including mutants or biologically active fragments thereof), a polysaccharide, a glycoprotein, a glycolipid, a nucleic acid, or a combination thereof.

In the present specification and claims, the term “antibody” is used in the broadest sense and covers fully assembled antibodies, antibody fragments that bind with antigens, such as antigen-binding fragment (Fab/Fab′), F(ab′)₂ fragment (having two antigen-binding Fab portions linked together by disulfide bonds), variable fragment (Fv), single chain variable fragment (scFv), bi-specific single-chain variable fragment (bi-scFv), nanobodies, unibodies and diabodies. “Antibody fragments” comprise a portion of an intact antibody, preferably the antigen-binding region or variable region of the intact antibody. Typically, an “antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The well-known immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, with each pair having one “light” chain (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V_(L)) and variable heavy chain (V_(H)) refer to these light and heavy chains, respectively. According to embodiments of the present disclosure, the antibody fragment can be produced by modifying the nature antibody or by de novo synthesis using recombinant DNA methodologies. In certain embodiments of the present disclosure, the antibody and/or antibody fragment can be bispecific, and can be in various configurations. For example, bispecific antibodies may comprise two different antigen binding sites (variable regions). In various embodiments, bispecific antibodies can be produced by hybridoma technique or recombinant DNA technique. In certain embodiments, bispecific antibodies have binding specificities for at least two different epitopes.

The term “specifically binds” as used herein, refers to the ability of an antibody or an antigen-binding fragment thereof, to bind to an antigen with a dissociation constant (Kd) of no more than about 1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, 1×10⁻¹² M, and/or to bind to an antigen with an affinity that is at least two-folds greater than its affinity to a nonspecific antigen.

The term “treatment” as used herein includes preventative (e.g., prophylactic), curative or palliative treatment; and “treating” as used herein also includes preventative (e.g., prophylactic), curative or palliative treatment. In particular, the term “treating” as used herein refers to the application or administration of the present molecular construct or a pharmaceutical composition comprising the same to a subject, who has a medical condition a symptom associated with the medical condition, a disease or disorder secondary to the medical condition, or a predisposition toward the medical condition, with the purpose to partially or completely alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of said particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition, and/or to a subject who exhibits only early signs of a disease, disorder and/or condition, for the purpose of decreasing the risk of developing pathology associated with the disease, disorder and/or condition.

The term “effective amount” as used herein refers to the quantity of the present molecular construct that is sufficient to yield a desired therapeutic response. An effective amount of an agent is not required to cure a disease or condition but will provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered or prevented, or the disease or condition symptoms are ameliorated. The effective amount may be divided into one, two, or more doses in a suitable form to be administered at one, two or more times throughout a designated time period. The specific effective or sufficient amount will vary with such factors as particular condition being treated, the physical condition of the patient (e.g., the patient's body mass, age, or gender), the type of subject being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives. Effective amount may be expressed, for example, as the total mass of active component (e.g., in grams, milligrams or micrograms) or a ratio of mass of active component to body mass, e.g., as milligrams per kilogram (mg/kg).

The terms “application” and “administration” are used interchangeably herein to mean the application of a molecular construct or a pharmaceutical composition of the present invention to a subject in need of a treatment thereof.

The terms “subject” and “patient” are used interchangeably herein and are intended to mean an animal including the human species that is treatable by the molecular construct, pharmaceutical composition, and/or method of the present invention. The term “subject” or “patient” intended to refer to both the male and female gender unless one gender is specifically indicated. Accordingly, the term “subject” or “patient” comprises any mammal, which may benefit from the treatment method of the present disclosure. Examples of a “subject” or “patient” include, but are not limited to, a human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird and fowl. In an exemplary embodiment, the patient is a human. The term “mammal” refers to all members of the class Mammalia, including humans, primates, domestic and farm animals, such as rabbit, pig, sheep, and cattle; as well as zoo, sports or pet animals; and rodents, such as mouse and rat. The term “non-human mammal” refers to all members of the class Mammalis except human.

The present disclosure is based, at least on the construction of the T-E pharmaceuticals that can be delivered to target cells, target tissues or organs at increased proportions relative to the blood circulation, lymphoid system, and other cells, tissues or organs. When this is achieved, the therapeutic effect of the pharmaceuticals is increased, while the scope and severity of the side effects and toxicity is decreased. It is also possible that a therapeutic effector is administered at a lower dosage in the form of a T-E molecule, than in a form without a targeting component. Therefore, the therapeutic effector can be administered at lower dosages without losing potency, while lowering side effects and toxicity.

Diseases that can Benefit from Better Drug Targeting

Drugs used for many diseases can be improved for better efficacy and safety, if they can be targeted to the disease sites, i.e., if they can be localized or partitioned to the disease sites more favorably than the normal tissues or organs. Certain antibody drugs, which target infectious microorganisms or their toxic products, can be improved, if they are empowered with the ability to recruit immunocytes, which phagocytose and clear the antibody-bound particles. Following are primary examples of diseases, in which drugs can be improved if they can be preferentially distributed to the disease sites or cells or if they can recruit phagocytic immunocytes.

Disease/Condition Associated with Blood Clot

There are two main aspects of pharmaceutical needs in dealing with the pathological problems of blood clotting (or coagulation): one aspect is to prevent or inhibit pathological blood clots to form or to grow in size once a nucleus of clot is formed, and the other aspect is to dissolve already-formed pathological clots timely. In both aspects, there are batteries of pharmaceutical products available clinically. Research aiming at developing still better products is continuing actively and a number of products are in clinical trials.

Patients suffered from various complications (e.g., those resulted from cardiovascular, endocrine, or other bodily regulatory conditions, surgical, the use of medicine, and other factor) have the tendency to develop blood clots. The clots may cause hemorrhagic strokes, head trauma, myocardial infarction, pulmonary embolism, or deep vein thrombosis, which often lead to serious, life threatening clinical conditions.

Coagulation involves a cascade of protease-catalyzed events, which amplify in sequence. Toward the later steps, Factor Xa cleaves prothrombin to thrombin, and thrombin cleaves fibrinogen to fibrin, which in combination with platelets forms the meshwork of a clot. The dissolution of the blood clot involves plasmin, which is generated from plasminogen via one of several of enzymes, including tissue plasminogen activator.

A. The Use of T-E Molecules for Preventing the Formation of a Blood Clot

A large number of indirect inhibitors of Factor Xa have been developed and used. For many decades, the inhibitors are primarily heparin, which is a mixture of naturally occurring polysaccharides of glycosaminoglycan of varying molecular weights from 5 to 30 kDa, low-molecular weight heparin, and heparinoid compounds. Those substances bind to heparin-binding proteins, including anti-thrombin, thus potentiating those substances to inhibit Factor Xa, thereby inhibiting the clot formation. Tissue factor pathway inhibitor (TFPI), a single-chain serum protein of 34,000-40,000 Daltons depending on the degree of proteolysis, can inhibit Factor Xa. However, it is not produced by recombinant DNA technology as a therapeutic.

A number of direct inhibitors of thrombin have also been developed and used clinically. Naturally recovered hirudin from medical leeches and recombinant hirudin, which bind to thrombin, were used for many years before they were discontinued because of the introduction of other better medicines.

More recently, several small molecules that are direct inhibitors of Factor Xa, or thrombin have been developed and approved for clinical uses in preventing coagulation in several clinical indications. In one set of clinical applications, these small molecules are direct inhibitors of Factor Xa, and may be apixaban, edoxaban, or rivaroxaban. In another set of applications, these small molecules are direct inhibitors of thrombin, and may be argatroban or dabigatran. Ximelagatran, a direct thrombin inhibitor, has favorable kinetics and may be administered in very small doses; however, it has been withdrawn from the market due to hepatoxicity problems. It is noted that Ximelagatran is a pro-drug, and the orally taken ximelagatran is converted in the liver to melagatran, which is the active form that binds to and inhibits thrombin. It is very possible that the reduced dosage of melagatran relative to that of ximelagatran and the avoidance of conversion of ximelagatran in the live can avoid the hepatoxicity seen with ximelagatran.

We rationalize that an effective approach to deal with clot formation is to inhibit a nucleus of a clot from growing into a pathological clot. Therefore, if an increased amount of Factor Xa inhibitor or thrombin inhibitor or both are brought to the clot nucleus, the clot will be prevented from growing in size and becoming pathological. We can use IgG or scFv of an anti-fibrin antibody to carry a drug bundle of an inhibitor of Factor Xa or an inhibitor of thrombin or combined bundles of both kinds of inhibitors to the newly formed nucleus of clot. Both the Factor Xa inhibitor molecules (such as, apixaban, edoxaban, and rivaroxaban) and the thrombin inhibitor molecules (such as, argatroban and melagatran) have an NH₂ group for conjugation with a linker unit proposed in the present disclosure.

Because the Factor Xa inhibitor and/or thrombin inhibitor are carried to the site of the clot by an anti-fibrin antibody, smaller amounts of the inhibitors than those used without anti-fibrin targeting will be required. Furthermore, because the blood stream flows in the blood vessels, the concentration effects of the carried inhibitors surrounding the clot will be significant only when the nucleus of the clot has grown to a certain size. Therefore, the physiologically required blood coagulation to mend minute internal wounds will not be affected. Thus, the targeting of Factor Xa and thrombin inhibitors by an anti-fibrin antibody to the clot should be therapeutically more effective, while reducing side effects of internal bleeding.

According to embodiments of the present disclosure, T-E molecules in the joint-linker configuration contain scFv specific for fibrin as targeting elements and a Factor Xa inhibitor (apixaban, edoxaban, or rivaroxaban) or thrombin inhibitor (argatroban and melagatran) or both as effector elements.

(B) The use of T-E molecules to speed up the dissolution of blood clots

To administer proper medications for dissolving pathological clots in a timely, tightly controlled fashion has been a very important pharmaceutical challenge. The development of several forms of recombinant human tissue plasminogen activator (tPA), including alteplase, reteplase, tenecteplase, and lanoteplase, has solved a significant part of the thrombosis problems. However, the use of tPA in many cases either is not sufficient to dissolve the clots or causes serious internal bleeding, or both. The controlled use of dosages and administration schedule of tPA is still an area of active research. The clinical studies comparing the several forms of recombinant tPA are also very active. From the wealth of published literature on tPA and its variants and their medical uses, it is apparent that the various properties of tPA, including the affinity in binding to fibrin, its half-life, the susceptibility to breakdown by liver cells, and the resistance to plasminogen activator inhibitor all play part in the desired properties of the tPA for a particular clinical condition.

The molecular structure of intact tPA is complex, comprising several structural domains with discrete functions or activities, although not all of these domains are required for a thrombolytic product suitable for use in dissolving blood clots. A full-length tPA molecule (alteplase) with 527 amino acid residues contains, (i) a fibronectin finger domain that binds to fibrin, (2) an epidermal growth factor domain that binds to hepatocytes and facilitates tPA's clearance, (3) a Kringle 1 domain that binds to hepatic endothelial cells and facilitates tPA' clearance, (4) a Kringle 2 domain that binds to fibrin and activates the serine protease, and (5) a protease domain that cleaves the plasminogen and is inhibited by plasminogen activator inhibitor type 1 (PAI-1). Alteplase, tenecteplase, and lanoteplase are produced in mammalian cells, CHO cells, and reteplase is produced in bacteria.

Reteplase, which is 355 residues in length, does not contain the fibronectin finger, epidermal growth factor domain, and Kringle 1 domain. Reteplase is produced in bacteria, and therefore it does not contain the posttranslational carbohydrate modification. While reteplase has a lower affinity for fibrin and its protease is not activated to the extent as in alteplase, reteplase has a plasma half-life of 14-18 minutes; in contrast, the half-life period of alteplase only lasts 3-4 minutes in plasma. Reteplase is administered to patients in boli, while alteplase is administered in a bolus followed by an infusion.

Tenecteplase has the entire length of 527 amino acid residues of alteplase, but has mutations at three sites. Threonine at 103 is replaced by asparagine to allow glycosylation modification, and asparagine at 117 is replaced by glutamine to eliminate glycosylation. These mutations inhibit the clearance of the molecule by liver cells. In addition, the four residues at 296-299 (i.e., lysine-histidine-arginine-arginine) are replaced by four alanine residues, thus increasing the resistance to PAI-1 by 80 times. Tenecteplase has a plasma half-life of 18 minutes.

In lanoteplase, the fibronectin finger and the epidermal growth factor domain are deleted and the asparagine at 117 is replaced by glutamine. The plasma half-life of lanoteplase is increased to 45 minutes, which improves administration procedures.

In various clinical trials, the overall therapeutic efficacies of the four forms of tPA are about equal, and each seems to fit better than others do in particular clinical conditions.

In the present invention, it is rationalized that a moderate increase in binding strength to fibrin and a moderate increase of plasma half-life over those exhibited by reteplase will increase the therapeutic properties of reteplase. These molecular constructs will allow more specificity for binding to clots and hence allowing lower doses and fewer side effects. If they cannot fit to all clinical conditions pertaining to dissolving blood clots, they can be applied to some of the conditions. Thus, a preferred embodiment of the present invention for an improved tPA for dissolving blood clots is that 1-3 scFv of an anti-fibrin antibody are employed as targeting elements and 1-2 copies reteplase are employed as effector elements in a joint-linkers configuration. In an alternative construct, reteplase is conjugated to the linker-unit via its C-terminal in order that the N-terminal Kringle 2 domain is flexible in contacting fibrin meshwork in the clot. The C-terminal is extended with a linker, such as (GGGGS)₂ and a cysteine residue. In still another embodiment, an Fc-fusion protein construct linking a tPA or its fragment and scFv specific for fibrin may also be applicable for facilitating the dissolution of pathological clots.

Part I Multi-Arm Linkers for Treating Specific Diseases

I-(i) Peptide Core for Use in Multi-Arm Linker

The first aspect of the present disclosure pertains to a linker unit that comprises, (1) a center core that comprises 2-15 lysine (K) residues, and (2) 2-15 linking arms respectively linked to the K residues of the center core. The present center core is characterized in having or being linked with an azide group, an alkyne group, a tetrazine group, or a strained alkyne group at its N- or C-terminus.

In the preparation of the present linker unit, a PEG chain having a N-hydroxysuccinimidyl (NHS) group at one terminus and a functional group (e.g., an NHS, a maleimide, an azide, an alkyne, a tetrazine, or a strained alkyne group) at the other terminus is linked to the K residue of the center core by forming an amide bond between the NHS group of the PEG chain and the amine group of the K residue. In the present disclosure, the PEG chain linked to the K residue is referred to as a linking arm, which has a functional group at the free-terminus thereof.

According to the embodiments of the present disclosure, the center core is a polypeptide that has 8-120 amino acid residues in length and comprises 2 to 15 lysine (K) residues, in which each K residue and the next K residue are separated by a filler sequence.

According to embodiments of the present disclosure, the filler sequence comprises glycine (G) and serine (S) residues; preferably, the filler sequence consists of 2-15 residues selected from G, S, and a combination thereof. For example, the filler sequence can be,

GS, GGS, GSG, (SEQ ID NO: 1) GGGS, (SEQ ID NO: 2) GSGS, (SEQ ID NO: 3) GGSG, (SEQ ID NO: 4) GSGGS, (SEQ ID NO: 5) SGGSG, (SEQ ID NO: 6) GGGGS, (SEQ ID NO: 7) GGSGGS, (SEQ ID NO: 8) GGSGGSG, (SEQ ID NO: 9) SGSGGSGS, (SEQ ID NO: 10) GSGGSGSGS, (SEQ ID NO: 11) SGGSGGSGSG, (SEQ ID NO: 12) GGSGGSGGSGS, (SEQ ID NO: 13) SGGSGGSGSGGS, (SEQ ID NO: 14) GGGGSGGSGGGGS, (SEQ ID NO: 15) GGGSGSGSGSGGGS,  or (SEQ ID NO: 16) SGSGGGGGSGGSGSG.

The filler sequence placed between two lysine residues may be variations of glycine and serine residues in somewhat random sequences and/or lengths. Longer fillers may be used for a polypeptide with fewer lysine residues, and shorter fillers for a polypeptide with more lysine residues. Hydrophilic amino acid residues, such as aspartic acid and histidine, may be inserted into the filler sequences together with glycine and serine. As alternatives for filler sequences made up with glycine and serine residues, filler sequences may also be adopted from flexible, soluble loops in common human serum proteins, such as albumin and immunoglobulins.

Basically, the filler sequences between lysine residues cover peptides with glycine and serine residues. However, they can alternatively be peptides composed of amino acids excluding one with amine group in its side chain. Those amino acids are predominantly, but not necessarily entirely hydrophilic amino acids. The amino acids are not necessarily naturally occurring amino acids.

According to certain preferred embodiments of the present disclosure, the center core comprises 2-15 units of the sequence of G₁₋₅SK. Alternatively, the polypeptide comprises the sequence of (GSK)₂₋₁₅; that is, the polypeptide comprises at least two consecutive units of the sequence of GSK. For example, the present center core may comprises the amino acid sequence of the following,

(SEQ ID NO: 17) Ac-CGGSGGSGGSKGSGSK, (SEQ ID NO: 18) Ac-CGGSGGSGGSKGSGSKGSK,  or (SEQ ID NO: 19) Ac-CGSKGSKGSKGSKGSKGSKGSKGSKGSKGSK, in which Ac represents the acetyl group.

According to certain embodiments of the present disclosure, the center core is a polypeptide that comprises the sequence of (X_(aa)-K)_(n), in which X_(aa) is a PEGylated amino acid having 2 to 12 repeats of ethylene glycol (EG) unit, and n is an integral from 2 to 15.

As would be appreciated, the lysine residue of the present center core may be substituted with an amino acid, which side chain contains an amine group. For example, an α-amino acid with (CH₂-)nNH₂ side chain, where n=1-3 or 5; an α-amino acid with (CH(OH)-)nCH₂—NH₂ side chain, where n=1-5; an α-amino acid with (CH₂—CH(OH)-)nCH₂—NH₂ side chain, where n=1-3; an α-amino acid with (CH₂—CH₂—O-)nCH₂—NH₂ side chain, where n=1-2. These amino acids are not necessarily naturally occurring amino acids.

As described above, the present center core is characterized in having or being linked with an azide group, an alkyne group, a tetrazine group, or a strained alkyne group at its N- or C-terminus. According to some embodiments of the present disclosure, the present center core comprises, at its N- or C-terminus, an amino acid residue having an azide group or an alkyne group. The amino acid residue having an azide group can be, L-azidohomoalanine (AHA), 4-azido-L-phenylalanine, 4-azido-D-phenylalanine, 3-azido-L-alanine, 3-azido-D-alanine, 4-azido-L-homoalanine, 4-azido-D-homoalanine, 5-azido-L-ornithine, 5-azido-d-ornithine, 6-azido-L-lysine, or 6-azido-D-lysine. For example, the present center core may have the sequence of,

-   -   Ac-(GSK)₂₋₇-(G₂₋₄S)₁₋₈-A^(AH),     -   Ac-A^(AH)-(SG₂₋₄)₁₋₈-(GSK)₂₋₇,     -   Ac-A^(AH)-(SG₂₋₄)₀₋₇-(GSK)₂₋₆-(G₂₋₄S)₁₋₈-C,     -   Ac-C-(SG₂₋₄)₀₋₇-(GSK)₂₋₆-(G₂₋₄S)₁₋₈-A^(AH),     -   Ac-K-(Xaa₂₋₁₂-K)₂₋₄-Xaa₂₋₁₂-A^(AH),     -   Ac-A^(AH)-Xaa₂₋₁₂-K-(Xaa₂₋₁₂-K)₂₋₄,     -   Ac-A^(AH)-Xaa₂₋₁₂-K-(Xaa₂₋₁₂-K)₁₋₃-Xaa₂₋₁₂-C, or     -   Ac-C-Xaa₂₋₁₂-K-(Xaa₂₋₁₂-K)₁₋₃-Xaa₂₋₁₂-A^(AH),         in which Xaa is a PEGylated amino acid having specified repeats         of EG unit, Ac represents the acetyl group, and A^(AH)         represents the AHA residue.

Exemplary amino acid having an alkyne group includes, but is not limited to, L-homopropargylglycine (L-HPG), D-homopropargylglycine (D-HPG), or beta-homopropargylglycine (β-HPG). In this case, the present center core may have the sequence of,

-   -   Ac-(GSK)₂₋₇-(G₂₋₄S)₁₋₈-G^(HP),     -   Ac-G^(HP)-(SG₂₋₄)₁₋₈-(GSK)₂₋₇,     -   Ac-G^(HP)-(SG₂₋₄)₀₋₇-(GSK)₂₋₆-(G₂₋₄S)₁₋₈-C,     -   Ac-C-(SG₂₋₄)₀₋₇-(GSK)₂₋₆-(G₂₋₄S)₁₋₈-G^(HP),     -   Ac-K-(Xaa₂₋₁₂-K)₂₋₄-Xaa₂₋₁₂-G^(HP),     -   Ac-C^(HP)-Xaa₂₋₁₂-K-(Xaa₂₋₁₂-K)₂₋₄,     -   Ac-C^(HP)-Xaa₂₋₁₂-K-(Xaa₂₋₁₂-K)₁₋₃-Xaa₂₋₁₂-C, or     -   Ac-C-Xaa₂₋₁₂-K-(Xaa₂₋₁₂-K)₁₋₃-Xaa₂₋₁₂-G^(HP),         in which Xaa is a PEGylated amino acid having specified repeats         of EG unit, Ac represents the acetyl group, and G^(HP)         represents the HPG residue.

It is noted that many of the amino acids containing an azide or alkyne group in their side chains and PEGylated amino acids are available commercially in t-boc (tert-butyloxycarbonyl)- or Fmoc (9-fluorenylmethyloxycarbonyl)-protected forms, which are readily applicable in solid-phase peptide synthesis.

According to some working examples of the present disclosure, the center core may comprise the sequence of,

(SEQ ID NO: 21) Ac-G^(HP)GGSGGSGGSKGSGSK, (SEQ ID NO: 22) Ac-G^(HP)GGSGGSGGSKGSGSKGSK, (SEQ ID NO: 23) Ac-A^(AH)GGSGGSGGSKGSGSKGSK, (SEQ ID NO: 24) Ac-G^(HP)GGSGGSGGSKGSGSKGSGSC, (SEQ ID NO: 25) Ac-C-Xaa₂-K-Xaa₂-K-Xaa₂-K, or (SEQ ID NO: 26) Ac-C-Xaa₆-K-Xaa₆-K-Xaa₆-K-Xaa₆-K-Xaa₆-K, in which Xaa is a PEGylated amino acid having specified repeats of EG unit, Ac represents the acetyl group, A^(AH) represents the AHA residue, and G^(HP) represents the HPG residue.

Alternatively, the present center core is linked with a coupling arm, which has a functional group (e.g., an azide group, an alkyne group, a tetrazine group, or a strained alkyne group) at the free-terminus thereof (that is, the terminus that is not linked to the center core). In these cases, the present center core comprises a cysteine residue at its N- or C-terminus. To prepare a linker unit linked with a coupling arm, a PEG chain having a maleimide group at one terminus and a functional group at the other terminus is linked to the cysteine residue of the center core via thiol-maleimide reaction occurred between the maleimide group of the PEG chain and the thiol group of the cysteine residue. In the present disclosure, the PEG chain linked to the cysteine residue of the center core is referred to as the coupling arm, which has a functional group at the free-terminus thereof.

As would be appreciated, the cysteine residue of the present center core may be substituted with an amino acid, which side chain contains a sulfhydryl group. For example, an α-amino acid with (CH(OH)-)nCH₂—SH side chain, where n=1-5; an α-amino acid with (CH₂—CH(OH)-)nCH₂—SH side chain, where n=1-3; an α-amino acid with (CH₂—CH₂—O-)nCH₂—SH side chain, where n=1-2. The amino acid is not necessarily naturally occurring amino acids. The cysteine residue need not be placed at the N- or C-terminal of the peptide core. For example, the cysteine residue can be placed in the middle of the peptide, so that the lysine residues are distributed on two sides of the cysteine residue.

Preferably, the coupling arm has a tetrazine group or a strained alkyne group (e.g., a cyclooctene or cyclooctyne group) at the free-terminus thereof. These coupling arms have 2-12 EG units. According to the embodiments of the present disclosure, the tetrazine group is 1,2,3,4-tetrazine, 1,2,3,5-tetrazine, 1,2,4,5-tetrazine, or derivatives thereof. The strained alkyne group may be a cyclooctene or a cyclooctyne group. According to the working examples of the present disclosure, the cyclooctene group is a trans-cyclooctene (TCO) group; example of cyclooctyne group includes, but is not limited to, dibenzocyclooctyne (DBCO), difluorinated cyclooctyne (DIFO), bicyclononyne (BCN), and dibenzocyclooctyne (DICO). According to some embodiments of the present disclosure, the tetrazine group is 6-methyl-tetrazine.

Example of the present center core configured to be linked with the coupling arm includes, but is not limited to,

-   -   Ac-(GSK)₂₋₇-(G₂₋₄S)₁₋₈-C-Xaa₂₋₁₂-tetrazine,     -   Ac-(GSK)₂₋₇-(G₂₋₄S)₁₋₈-C-Xaa₂₋₁₂-strained alkyne,     -   Ac-K-(Xaa₂₋₁₂-K)₂₋₄-Xaa₂₋₁₂-C-Xaa₂₋₁₂-tetrazine,     -   Ac-K-(Xaa₂₋₁₂-K)₂₋₄-Xaa₂₋₁₂-C-Xaa₂₋₁₂-strained alkyne,     -   Tetrazine-Xaa₂₋₁₂-C(Ac)-(SG₂₋₄)₁₋₈-(GSK)₂₋₇,     -   Strained alkyne-Xaa₂₋₁₂-C(Ac)-(SG₂₋₄)₁₋₈-(GSK)₂₋₇,     -   Tetrazine-Xaa₂₋₁₂-C(Ac)-Xaa₂₋₁₂-K-(Xaa₂₋₁₂-K)₂₋₄, and     -   Strained alkyne-Xaa₂₋₁₂-C(Ac)-Xaa₂₋₁₂-K-(Xaa₂₋₁₂-K)₂₋₄.

Alternatively, the center core has an azide or alkyne group at one terminus and a coupling arm with tetrazine or strained alkyne group at the other terminus. Examples are the following:

-   -   Ac-A^(AH)-(SG₂₋₄)₀₋₇-(GSK)₂₋₆-(G₂₋₄S)₁₋₈-C-Xaa₂₋₁₂-tetrazine,     -   Ac-A^(AH)-(SG₂₋₄)₀₋₇-(GSK)₂₋₆-(G₂₋₄S)₁₋₈-C-Xaa₂₋₁₂-strained         alkyne,     -   Tetrazine-Xaa₂₋₁₂-C(Ac)-(SG₂₋₄)₀₋₇-(GSK)₂₋₆-(G₂₋₄S)₁₋₈-A^(AH),     -   Strained         alkyne-Xaa₂₋₁₂-C(Ac)-(SG₂₋₄)₀₋₇-(GSK)₂₋₆-(G₂₋₄S)₁₋₈-A^(AH),     -   Ac-A^(AH)-Xaa₂₋₁₂-K-(Xaa₂₋₁₂-K)₁₋₃-Xaa₂₋₁₂-C-Xaa₂₋₁₂-tetrazine,     -   Ac-A^(AH)-Xaa₂₋₁₂-K-(Xaa₂₋₁₂-K)₁₋₃-Xaa₂₋₁₂-C-Xaa₂₋₁₂-strained         alkyne,     -   Tetrazine-Xaa₂₋₁₂-C(Ac)-Xaa₂₋₁₂-K-(Xaa₂₋₁₂-K)₁₋₃-Xaa₂₋₁₂-A^(AH),     -   Strained         alkyne-Xaa₂₋₁₂-C(Ac)-Xaa₂₋₁₂-K-(Xaa₂₋₁₂-K)₁₋₃-Xaa₂₋₁₂-A^(AH),     -   Ac-C^(HP-)(SG₂₋₄)₀₋₇-(GSK)₂₋₆-(G₂₋₄S)₁₋₈-C-Xaa₂₋₁₂-tetrazine,     -   Ac-G^(HP)-(SG₂₋₄)₀₋₇-(GSK)₂₋₆-(G₂₋₄S)₁₋₈-C-Xaa₂₋₁₂-strained         alkyne,     -   Tetrazine-Xaa₂₋₁₂-C(Ac)-(SG₂₋₄)₀₋₇-(GSK)₂₋₆-(G₂₋₄S)₁₋₈-G^(HP),     -   Strained         alkyne-Xaa₂₋₁₂-C(AC)-(SG₂₋₄)₀₋₇-(GSK)₂₋₆-(G₂₋₄S)₁₋₈-G^(HP),     -   Ac-G^(HP)-Xaa₂₋₁₂-K-(Xaa₂₋₁₂-K)₁₋₃-Xaa₂₋₁₂-C-Xaa₂₋₁₂-tetrazine,     -   Ac-G^(HP)-Xaa₂₋₁₂-K-(Xaa₂₋₁₂-K)₁₋₃-Xaa₂₋₁₂-C-Xaa₂₋₁₂-strained         alkyne,     -   Tetrazine-Xaa₂₋₁₂-C(Ac)-Xaa₂₋₁₂-K-(Xaa₂₋₁₂-K)₁₋₃-Xaa₂₋₁₂-G^(HP),         and     -   Strained         alkyne-Xaa₂₋₁₂-C(Ac)-Xaa₂₋₁₂-K-(Xaa₂₋₁₂-K)₁₋₃-Xaa₂₋₁₂-G^(HP).

The polypeptide may also be synthesized using recombinant technology by expressing designed gene segments in bacterial or mammalian host cells. It is preferable to prepare the polypeptide as recombinant proteins if the core has high numbers of lysine residues with considerable lengths. As the length of a polypeptide increases, the number of errors increases, while the purity and/or the yield of the product decrease, if solid-phase synthesis was adopted. To produce a polypeptide in bacterial or mammalian host cells, a filler sequence ranges from a few amino acid residues to 10-20 residues may be placed between two K residues. Further, since AHA and HPG are not natural amino acids encoded by the genetic codes, the N-terminal or C-terminal residue for those recombinant polypeptides is cysteine. After the recombinant proteins are expressed and purified, the terminal cysteine residue is then reacted with short bifunctional cross-linkers, which have maleimide group at one end, which reacts with SH group of cysteine residue, and alkyne, azide, tetrazine, or strained alkyne at the other end.

The synthesis of a polypeptide using PEGylated amino acids involves fewer steps than that with regular amino acids such as glycine and serine resides. In addition, PEGylated amino acids with varying lengths (i.e., numbers of repeated ethylene glycol units) may be employed, offering flexibility for solubility and spacing between adjacent amino groups of lysine residues. Other than PEGylated amino acids, the center cores may also be constructed to comprise artificial amino acids, such as D-form amino acids, homo-amino acids, N-methyl amino acids, etc. Preferably, the PEGylated amino acids with varying lengths of polyethylene glycol (PEG) are used to construct the center core, because the PEG moieties contained in the amino acid molecules provide conformational flexibility and adequate spacing between conjugating groups, enhance aqueous solubility, and are generally weakly immunogenic. The synthesis of PEGylated amino acid-containing center core is similar to the procedures for the synthesis of regular polypeptides.

Optionally, for stability purpose, the present center core has an acetyl group to block the amino group at its N-terminus.

As could be appreciated, the number of the linking arms linked to the center core is mainly determined by the number of lysine resides comprised in the center core. Since there are at least two lysine residues comprised in the present center core, the present linker unit may comprise a plurality of linking arms.

Reference is now made to FIG. 1A. As illustrated, the linker unit 10A comprises a center core 11 a comprising one HPG (G^(HP)) residue and four lysine (K) residues respectively separated by filler sequences (denoted by the dots throughout the drawings). The filler sequences between the HPG residue and K residue or between any two K residues may comprise the same or different amino acid sequences. In this example, four linking arms 20 a-20 d are linked to the lysine residues by forming an amide linkage between the NHS group and the amine group of the lysine residue, respectively. As could be appreciated, certain features discussed above regarding the linker unit 10A or any other following linker units are common to other linker units disclosed herein, and hence some or all of these features are also applicable in the following examples, unless it is contradictory to the context of a specific embodiment. However, for the sake of brevity, these common features may not be explicitly repeated below.

FIG. 1B provides a linker unit 10B according to another embodiment of the present disclosure. The center core 11 b comprises one cysteine (C) residue and six lysine (K) residues respectively separated by the filler sequences. In this example, the linker unit 10B comprises six linking arms 20 a-20 f that are respectively linked to the lysine residues.

According to the embodiments of the present disclosure, the linking arm is a PEG chain having 2-20 repeats of EG units.

Unlike the linker unit 10A of FIG. 1A, the linker unit 1B further comprises a coupling arm 60. As discussed above, a PEG chain having a maleimide group at one end and a functional group at the other end is used to form the coupling arm 60. In this way, the coupling arm 60 is linked to the cysteine residue of the center core 11 b via thiol-maleimide reaction. In this example, the functional group at the free terminus of the coupling arm 60 is a tetrazine group 72. According to the embodiments of the present disclosure, the coupling arm is a PEG chain having 2-12 repeats of EG units.

When the release of effector elements at the targeted site is required, a cleavable bond can be installed in the linking arm. Such a bond is cleaved by acid/alkaline hydrolysis, reduction/oxidation, or enzymes. One embodiment of a class of cleavable PEG chains that can be used to form the coupling arm is NHS-PEG₂₋₂₀-S—S-maleimide, where S—S is a disulfide bond that can be slowly reduced, while the NHS group is used for conjugating with the amine group of the center core, thereby linking the PEG chain onto the center core. The maleimide group at the free terminus of the linking arm may be substituted by an azide, alkyne, tetrazine, or strained alkyne group.

According to the embodiments of the present disclosure, the linking arm linked to the K residue of the center core has a functional group (i.e., a maleimide, an NHS, an azide, an alkyne, a tetrazine, or a strained alkyne group) at its free terminus. Preferably, when the free terminus of the linking arm is an azide, alkyne, or cyclooctyne group, then the amino acid residue at the N- or C-terminus of the center core is a cysteine residue, and the free terminus of the coupling arm is a tetrazine or cyclooctene group. Alternatively, when the free terminus of the linking arm is a tetrazine group or cyclooctene group, then the amino acid residue at the N- or C-terminus of the center core has an azide or alkyne group, or the amino acid residue at the N- or C-terminus of the center core is a cysteine residue, and the free terminus of the coupling arm is an azide, the alkyne, or the cyclooctyne group

Depending on the functional group (i.e., a maleimide, an NHS, an azide, an alkyne, a tetrazine, or a strained alkyne group) present at the free terminus of the linking arm, it is feasible to design a functional element (such as, a targeting element, an effector element, or an element for improving the pharmacokinetic property) with a corresponding functional group, so that the functional element may linked to the free terminus of the linking arm via any of the following chemical reactions,

(1) forming an amide bond therebetween: in this case, the linking arm has an NHS group at the free terminus, and the functional element has an amine group;

(2) the thiol-maleimide reaction: in this case, the linking arm has a maleimide group at the free terminus, and the functional element has an thiol group;

(3) the Copper(I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC reaction, or the “click” reaction for short): one of the free terminus of the linking arm and the functional element has an azide group, while the other has an alkyne group; the CuAAC reaction is exemplified in Scheme 1;

(4) the inverse electron demand Diels-Alder (iEDDA) reaction: one of the free terminus of the linking arm and the functional element has a tetrazine group, while the other has a cyclooctene group; the iEDDA reaction is exemplified in Scheme 2; or

(5) the strained-promoted azide-alkyne click chemistry (SPAAC) reaction: one of the free terminus of the linking arm and the functional element has an azide group, while the other has an cyclooctyne group; the SPAAC reaction is exemplified in Scheme 3.

The CuAAC reaction yields 1,5 di-substituted 1,2,3-triazole. The reaction between alkyne and azide is very selective and there are no alkyne and azide groups in natural biomolecules. Furthermore, the reaction is quick and pH-insensitive. It has been suggested that instead of using copper (I), such as cuprous bromide or cuprous iodide, for catalyzing the click reaction, it is better to use a mixture of copper (II) and a reducing agent, such as sodium ascorbate to produce copper (I) in situ in the reaction mixture. Alternatively, the second element can be linked to the N- or C-terminus of the present center core via a copper-free reaction, in which pentamethylcyclopentadienyl ruthenium chloride complex is used as the catalyst to catalyze the azide-alkyne cycloaddition.

For the sake of illustration, the functional elements linked to the linking arms are referred to as the first elements. As could be appreciated, the number of the first elements carried by the present linker unit depends on the number of K residues of the center core (and thus, the number of the linking arms). Accordingly, one of ordinary skill in the art may adjust the number of the first elements of the linker unit as necessary, for example, to achieve the desired targeting or therapeutic effect.

An example of a linker unit 10C having the first elements is illustrated FIG. 1C. Other than the features disused hereafter, FIG. 1C is quite similar to FIG. 1B. First, there are five K residues in the center core 11 d, and accordingly, five linking arms 20 a-20 e are linked thereto, respectively. Second, the linker unit 10C has five first elements 30 a-30 e linked to each of the linking arms 20 a-20 e. As disused below, the optional tetrazine group 72 allows for the conjugation with an additional functional element, another molecular construct (see, Part II or Part III below).

In order to increase the intended or desired effect (e.g., the therapeutic effect), the present linker unit may further comprise a second element in addition to the first element. For example, the second element can be either a targeting element or an effector element. In optional embodiments of the present disclosure, the first element is an effector element, while the second element may be another effector element, which works additively or synergistically with or independently of the first element. Still optionally, the first and second elements exhibit different properties; for example, the first element is a targeting element, and the second element is an effector element, and vice versa. Alternatively, the first element is an effector element, and the second element is an element capable of improving the pharmacokinetic property of the linker unit, such as solubility, clearance, half-life, and bioavailability. The choice of a particular first element and/or second element depends on the intended application in which the present linker unit (or multi-arm linker) is to be used. Examples of these functional elements are discussed below in Part I-(iii) of this specification.

Structurally, the second element is linked to the azide, alkyne, tetrazine, or strained alkyne group at the N- or C-terminus of the center core. Specifically, the second element may be optionally conjugated with a short PEG chain (preferably having 2-12 repeats of EG units) and then linked to the N- or C-terminal amino acid residue having an azide group or an alkyne group (e.g., AHA residue or HPG residue). Alternatively, the second element may be optionally conjugated with the short PEG chain and then linked to the coupling arm of the center core.

According to some embodiments of the present disclosure, the center core comprises an amino acid having an azide group (e.g., the AHA residue) at its N- or C-terminus; and accordingly, a second element having an alkyne group is linked to the N- or C-terminus of the center core via the CuAAC reaction. According to other embodiments of the present disclosure, the center core comprises an amino acid having an alkyne group (e.g., the HPG residue) at its N- or C-terminus; and a second element having an azide group is thus capable of being linked to the N- or C-terminus of the center core via the CuAAG reaction.

FIG. 1D provides an example of the present linker unit 10D carrying a plurality of first elements and one second element. In this example, the center core 11 c comprises one HPG (G^(HP)) residue and five lysine (K) residues. Five linking arms 20 a-20 e are respectively linked to the five K residues of the center core 11 c; and five first elements 30 a-30 e are respectively linked to said five linking arms 20 a-20 e via the thiol-maleimide reaction. In addition to the first elements, the linker unit 10D further comprises one second element 50 that is linked to one end of a short PEG chain 62. Before being conjugated with the center core 11 c, the other end of the short PEG chain 62 has an azide group. In this way, the azide group may react with the HPG residue that having an alkyne group via CuAAC reaction, so that the second element 50 is linked to the center core 11 c. The solid dot 40 depicted in FIG. 1D represents the chemical bond resulted from the CuAAC reaction occurred between the HPG residue and the azide group.

Alternatively, the second element is linked to the center core via a coupling arm. According to certain embodiments of the present disclosure, the coupling arm has a tetrazine group, which can be efficiently linked to a second element having a TCO group via the iEDDA reaction. According to other embodiments of the present disclosure, the coupling arm has a TCO group, which is capable of being linked to a second element having a tetrazine group via the iEDDA reaction. In the iEDDA reaction, the strained cyclooctene that possess remarkably decreased activation energy in contrast to terminal alkynes is employed, and thus eliminates the need of an exogenous catalyst.

Reference is now made to FIG. 1E, in which the center core 11 d of the linker unit 10E comprises a terminal cysteine (C) residue and five lysine (K) residues. As depicted in FIG. 1E, five linking arms 20 a-20 e are respectively linked to the five K residue of the center core 11 d, and then five first elements 30 a-30 e are respectively linked to the five linking arms 20 a-20 e via thiol-maleimide reactions. The cysteine residue is linked to the coupling arm 60, which, before being conjugated with the second element, comprises a tetrazine group or a TCO group at its free-terminus. In this example, a second element 50 linked with a short PEG chain 62 having a corresponding TCO or tetrazine group can be linked to the coupling arm 60 via the iEDDA reaction. The ellipse 70 as depicted in FIG. 1E represents the chemical bond resulted from the iEDDA reaction occurred between the coupling arm 60 and the short PEG chain 62.

According to other embodiments of the present disclosure, before the conjugation with a second element, the coupling arm has an azide group. As such, the coupling arm can be linked to the second element having a strained alkyne group (e.g., the DBCO, DIFO, BCN, or DICO group) at the free-terminus of a short PEG chain via SPAAC reaction (see, scheme 3), and vice versa.

Reference is now made to FIG. 1F, in which the linker unit 10F has a structure similar to the linker unit 10E of FIG. 1E, except that the coupling arm 60 comprises an azide or a strained alkyne group (e.g., the DBCO, DIFO, BCN, or DICO group), instead of the tetrazine or TCO group. Accordingly, the second element 50 linked with a short PEG chain 62 may have a corresponding strained alkyne (e.g., DBCO, DIFO, BCN, or DICO) or azide group, so that it can be linked to the coupling arm 60 via the SPAAC reaction. The diamond 90 as depicted in FIG. 1F represents the chemical bond resulted from the SPAAC reaction occurred between the coupling arm 60 and the short PEG chain 62.

Scheme 4 is an exemplary illustration of the process of preparing the present linker unit.

In step 1, the center core comprising the amino acid sequence of (GSK)₃ and a L-azidohomoalanine (AHA) residue at the C-terminus thereof is prepared. In step 2, three linking arms are respectively linked to the lysine (K) residues of the center core via forming an amide bond between the NHS group and the amine group; the linking arm linked to the center core has a maleimide (Mal) group at the free-terminus thereof. In step 3, three anti-fibrin scFvs (scFv α fibrin) as the first element are respectively linked to the linking arms via the thiol-maleimide reaction. Meanwhile, in step 4, one tPA analogue as the second element is linked with a short PEG chain that has 4 repeats of EG units and a DBCO group at the free terminus. Finally, in step 5, the second element is linked to the AHA residue of the center core via the SPAAC reaction.

Scheme 5 illustrates another example of the process for preparing the present linker unit.

In step 1, the center core comprising the amino acid sequence of (K-Xaa)₃ and a cysteine residue at the C-terminus thereof is prepared. In step 2, a PEG chain (as the coupling arm) that has the maleimide (Mal) group at one terminus and a tetrazine group at the other terminus is linked to the cysteine residue via the thiol-maleimide reaction. Then, in step 3, three linking arm are respectively linked to the lysine (K) residues of the center core. Next, three anti-fibrin scFvs (scFv α fibrin) as the first elements are respectively linked to the linking arms via the thiol-maleimide reaction as described in step 4. Meanwhile, in step 5, one tPA analogue as the second element is linked with a short PEG chain that has 3 repeats of EG units and a TCO group at the free terminus. Finally, in step 6, the second element is linked to the coupling arm via the iEDDA reaction.

PEGylation is a process, in which a PEG chain is attached or linked to a molecule (e.g., a drug or a protein). It is known that PEGylation imparts several significant pharmacological advantages over the unmodified form, such as improved solubility, increased stability, extended circulating life, and decreased proteolytic degradation. According to one embodiment of the present disclosure, the second element is a PEG chain, which has a molecular weight of about 20,000 to 50,000 Daltons.

FIG. 1G provides an alternative example of the present linker unit (linker unit 10G), in which five first elements 30 are respectively linked to the lysine residues via the linking arms 20, and the AHA (A^(AH)) residue of the center core 11 e is linked with a PEG chain 80 via the CuAAC reaction. The solid dot 40 depicted in FIG. 1G represents the chemical bond resulted from the CuAAC reaction occurred between the AHA residue and the PEG chain 80.

FIG. 1H provides another example of the present disclosure, in which the N-terminus of the center core 11 d is a cysteine residue that is linked to a coupling arm 60. A PEG chain 80 can be efficiently linked to the coupling arm 60 via the iEDDA reaction. The ellipse 70 of the linker unit 10H represents the chemical bond resulted from the iEDDA reaction occurred between the coupling arm 60 and the PEG chain 80.

FIG. 11 provides an alternative example of the present linker unit, in which the linker unit 101 has a structure similar to the linker unit 10G of FIG. 1 G, except that the PEG chain 80 is linked to the coupling arm 60 via the SPAAC reaction. The diamond 90 depicted in FIG. 11 represents the chemical bond resulted from the SPAAC reaction occurred between the coupling arm 60 and the PEG chain 80.

According to some embodiments of the present disclosure, in addition to the first and second elements, the present linker unit further comprises a third element. In this case, one of the N- and C-terminus of the center core is an amino acid having an azide group or an alkyne group, while the other of the N- and C-terminus of the center core is a cysteine residue. The lysine residues of the center core are respectively linked with the linking arms, each of which has a maleimide group at its free terminus; whereas the cysteine residue of the center core is linked with the coupling arm, which has a tetrazine group or a strained alkyne group at its free terminus. As described above, the first element is therefore linked to the linking arm via the thiol-maleimide reaction, and the second element is linked to the coupling arm via the iEDDA reaction. Further, a third element is linked to the terminal amino acid having an azide group or an alkyne group via the CuAAC reaction or SPAAC reaction.

Reference is now made to the linker unit 10J of FIG. 1J, in which the center core 11 f has an HPG (G^(HP)) residue at the N-terminus thereof and a cysteine residue at the C-terminus thereof. The linking arms 20 and the coupling arm 60 are respectively linked to the lysine (K) residues and the cysteine (C) residue of the center core 11 f. Further, five first elements 30 are respectively linked to the five linking arms 20, the second element (i.e., the PEG chain) 80 is linked to the coupling arm 60 via the short PEG chain 62, and the third element 50 is linked to the HPG residue. The solid dot 40 indicated the chemical bond resulted from the CuAAC reaction occurred between the HPG residue and the short PEG chain 62; while the ellipse 70 represents the chemical bond resulted from the iEDDA reaction occurred between the coupling arm 60 and the PEG chain 80.

FIG. 1K provides another embodiment of the present disclosure, in which the linker unit 10K has the similar structure with the linker unit 10J of FIG. 1J, except that the short PEG chain 62 is linked with the HPG residue via the SPAAC reaction, instead of the iEDDA reaction. The diamond 90 in FIG. 1K represents the chemical bond resulted from the SPAAC reaction occurred between the short PEG chain 62 and the HPG residue.

In the preferred embodiments of this disclosure, the linking arms have a maleimide group in the free terminus for conjugating with first elements having the sulfhydryl group via the thiol-maleimide reaction. Also, there is one cysteine residue or an amino acid residue with an azide or alkyne group at a terminus of the peptide core for attaching a coupling arm for linking a second element.

It is conceivable for those skilled in the arts that variations may be made. A conjugating group, other than maleimide, such as azide, alkyne, tetrazine, or strained alkyne may be used for the free terminus of the linking arms, for linking with first elements with a CuAAC, iEDDA, or SPAAC reaction. Also the cysteine residue (or an amino acid residue with an azide or alkyne group) of the peptide core needs not to be at the N- or C-terminus. Furthermore, two or more of such residues may be incorporated in the peptide core to attach multiple coupling arms for linking a plural of second elements.

I-(ii) Compound Core for Use in Multi-Arm Linker

In addition to the linker unit described in part I-(i) of the present disclosure, also disclosed herein is another linker unit that employs a compound, instead of the polypeptide, as the center core. Specifically, the compound is benzene-1,3,5-triamine, 2-(aminomethyl)-2-methylpropane-1,3-diamine, tris(2-aminoethyl)amine, benzene-1,2,4,5-tetraamine, 3,3′,5,5′-tetraamine-1,1′-biphenyl, tetrakis(2-aminoethyl)methane, tetrakis(ethylamine)hydrazine, N,N,N′,N′,-tetrakis(aminoethyl)ethylenediamine, benzene-1,2,3,4,5,6-hexaamine, 1-N,1-N,3-N,3-N,5-N,5-N-hexakis(methylamine)-benzene-1,3,5-triamine, 1-N,1-N,2-N,2-N,4-N,4-N,5-N,5-N,-octakis(methylamine)-benzene-1,2,4,5-triamine, benzene-1,2,3,4,5,6-hexaamine, or N,N-bis[(1-amino-3,3-diaminoethyl)pentyl]-methanediamine. Each of these compounds has 3 or more amine groups in identical or symmetrical configuration. Therefore, when one of the amine groups of a compound is conjugated with a coupling arm, all of the molecules of the compound have the same configuration.

Similar to the mechanism of linkage described in Part I-(i) of the present disclosure, each compound listed above comprises a plurality of amine groups, and thus, a plurality of PEG chains having NHS groups can be linked to the compound via forming an amine linkage between the amine group and the NHS group; the thus-linked PEG chain is designated as linking arm, which has a functional group (e.g., an NHS, a maleimide, an azide, an alkyne, a tetrazine, a cyclooctene, or a cyclooctyne group) at the free-terminus thereof. Meanwhile, at least one of the amine groups of the compound core is linked to another PEG chain, which has an NHS group at one end, and a functional group (e.g., an azide, alkyne, tetrazine, cyclooctene, or cycloodyne group) at the other end; the thus-linked PEG chain is designated as coupling arm, which has a functional group at the free-terminus thereof.

Accordingly, a first element can be linked to the linking arm via (1) forming an amide bond therebetween, (2) the thiol-maleimide reaction, (3) the CuAAC reaction, (4) the iEDDA reaction, or (5) SPAAC reaction. Meanwhile, the second element can be linked to the coupling arm via the CuAAC, iEDDA, or SPAAC reaction.

According to some embodiments of the present disclosure, the linking arm is a PEG chain having 2-20 repeats of EG units; and the coupling arm is a PEG chain having 2-12 repeats of EG unit.

Schemes 6 and 7 respectively depict the linkages between the center compound core and the linking arm, as well as the coupling arm. In schemes 6 and 7, “NHS” represents the NHS ester, “Mal” represents the maleimide group, “azide” represents the azide group, and “alkyne” represents the alkyne group.

The requirement of having multiple NH₂ groups exist in a symmetrical and identical orientation in the compound serving as the center core is for the following reason: when one of the NH₂ group is used for connecting a bifunctional linker arm with N-hydroxysuccinimidyl (NHS) ester group and alkyne, azide, tetrazine, or strained alkyne group, the product, namely, a core with a coupling arm having alkyne, azide, tetrazine or strained alkyne, is homogeneous and may be purified. Such a product can then be used to produce multi-arm linker units with all other NH₂ groups connected to linking arms with maleimide or other coupling groups at the other ends. If a compound with multiple NH₂ groups in non-symmetrical orientations, the product with one bifunctional linking arm/coupling arms is not homogeneous.

Some of those symmetrical compounds can further be modified to provide center cores with more linking arms/coupling arms. For example, tetrakis(2-aminoethyl)methane, which can be synthesized from common compounds or obtained commercially, may be used as a core for constructing linker units with four linking arms/coupling arms. Tetrakis(2-aminoethyl)methane can react with bis(sulfosuccinimidyl)suberate to yield a condensed product of two tetrakis(2-aminoethyl)methane molecules, which can be used as a core for constructing linker units having six linking arms/coupling arms. The linker units, respectively having three linking arms/coupling arms, four linking arms/coupling arms and six linking arms/coupling arms, can fulfill most of the need for constructing targeting/effector molecules with joint-linker configuration.

As would be appreciated, the numbers of the linking arm and/or the coupling arm and the element linked thereto may vary with the number of amine groups comprised in the center core. In some preferred embodiments, the numbers of the linking arm/coupling arm and the corresponding linking element linked thereto ranges from about 1-7.

Reference is now made to FIG. 2, in which benzene-1,2,4,5-tetraamine having 4 amine groups is depicted. Three of the amine groups are respectively linked to the linking arms 20, and one of the amine group is linked to the coupling arm 60, which has an azide group at its free-terminus. Three first elements 30 are then respectively linked to the three linking arms 20 via the thiol-maleimide reactions, and one second element 50 is linked to the coupling arm 60 via the CuAAC reaction. The solid dot 40 as depicted in FIG. 2 represents the chemical bond resulted from the CuAAC reaction occurred between the coupling arm 60 and the second element 50.

I-(iii) Functional Elements Suitable for Use in Multi-Arm Linker

In the case where the linker unit (or multi-arm linker) comprises only the first element but not the second and/or third element(s), the first element is an effector element that may elicit a therapeutic effect in a subject. On the other hand, when the present linker unit comprises elements in addition to first element(s), then at least one of the elements is an effector element, while the other may be another effector element, a targeting element, or an element capable of enhancing one or more pharmacokinetic properties of the linker unit (e.g., solubility, clearance, half-life, and bioavailability). For example, the linker unit may have two different kinds of effector element, one effector element and one targeting element or one pharmacokinetic property-enhancing element, two different kinds of targeting elements and one kind of effector element, two different kinds of effector elements and one kind of targeting element, or one kind of targeting element, one kind of effector element and one element capable of improving the pharmacokinetic property of the linker unit.

According to some embodiments of the present disclosure, the present linker unit is useful in preventing the formation of blood clot. In these embodiments, the present linker unit comprises the first element of an scFv specific for fibrin as the targeting element, and the second element of Factor Xa inhibitors or thrombin inhibitors as the effector element.

Preferably, the inhibitor of Factor Xa is selected from the group consisting of, apixaban, edoxaban, and rivaroxaban; and the inhibitor of thrombin is argatroban or melagatran.

Linker units for use in the treatment of thrombosis may comprise the first element of an scFv specific for fibrin as the targeting element, and the second element of tissue plasminogen activators (such as alteplase, reteplase, tenecteplase and lanoteplase) used as the effector element.

1-(iv) Use of Multi-Arm Linker

The present disclosure also pertains to method for treating various diseases using the suitable linker unit. Generally, the method comprises the step of administering to a subject in need of such treatment an effective amount of the linker unit according to embodiments of the present disclosure.

Compared with previously known therapeutic constructs, the present linker unit discussed in Part I is advantageous in two points:

(1) The number of the functional elements may be adjusted in accordance with the needs and/or applications. The present linker unit may comprise two elements (i.e., the first and second elements) or three elements (i.e., the first, second, and third elements) in accordance with the requirements of the application (e.g., the disease being treated, the route of administration of the present linker unit, and the binding avidity and/or affinity of the antibody carried by the present linker unit). For example, when the present linker unit is directly delivered into the tissue/organ (e.g., the treatment of eye), one element acting as the effector element may be enough, thus would eliminate the need of a second element acting as the targeting element. However, when the present linker unit is delivered peripherally (e.g., oral, enteral, nasal, topical, transmucosal, intramuscular, intravenous, or intraperitoneal injection), it may be necessary for the present linker unit to simultaneously comprise a targeting element that specifically targets the present linker unit to the lesion site; and an effector element that exhibits a therapeutic effect on the lesion site. For the purpose of increasing the targeting or treatment efficacy or increasing the stability of the present linker unit, a third element (e.g., a second targeting element, a second effector element, or a PEG chain) may be further included in the present linker unit.

(2) The first element is provided in the form of a bundle. As described above, the number of the first element may vary with the number of lysine residue comprised in the center core. If the number of lysine residue in the center core ranges from 2 to 15, then at least two first elements may be comprised in each linker unit. Thus, instead of providing one single molecule (e.g., cytotoxic drug and antibody) as traditional therapeutic construct or method may render, the present linker unit is capable of providing more functional elements (either as targeting elements or as effector elements) at one time, thereby greatly improves the therapeutic effect.

In certain therapeutic applications, it is desirable to have a single copy of a targeting or effector element. For example, a single copy of a targeting element can be used to avoid unwanted effects due to overly tight binding. This consideration is relevant, when the scFv has a relatively high affinity for the targeted antigen and when the targeted antigen is a cell surface antigen on normal cells, which are not targeted diseased cells. As an example, in using scFv specific for CD3 or CD16a to recruit T cells or NK cells to kill targeted cells, such as thyroid gland cells in patients with Grave's disease, a single copy of the scFv specific for CD3 or CD16a is desirable, so that unwanted effects due to cross-linking of the CD3 or CD16a may be avoided. Similarly, in using scFv specific for CD32 or CD16b to recruit phagocytic neutrophils and macrophages to clear antibody-bound viral or bacterial particles or their products, a single copy of scFv may be desirable. Also, in using scFv specific for transferrin receptor to carry effector drug molecules to the BBB for treating CNS diseases, a single copy of scFv specific for transferrin receptor is desirable. In still another example, it is desirable to have only one copy of long-chain PEG for enhancing pharmacokinetic properties. Two or more long PEG chains may cause tangling and affect the binding properties of the targeting or effector elements.

Part II Fc-Based Molecular Constructs for Preventing the Formation of Blood Clot and Treating Thrombosis and Uses Thereof

In the broad sense of the Fc-based configuration, immunoglobulin antibody can serve as the base of a targeting or effector element, and its corresponding effector or targeting element can be incorporated at the C-terminal of its two heavy γ chains in the form of scFv domains. For a typical “Fc-based” configuration, two-chain IgG.Fc is used as the base of the molecular platform. Each of the polypeptide chain is fused with one or two targeting and one or two effector elements, for a total of two to three elements on each chain. The T-E molecule with an Fc-based configuration will have a total of four to six elements (e.g., scFv, growth factor, or cytokines). Optionally, the Fc portion of the molecular constructs also carries Fc-mediated effector functions, ADCC, and/or complement-mediated activation. While in certain other applications, such Fc-mediated effector functions are avoided.

By selecting the T-E elements of the present Fc-based molecular construct, the molecular construct can be used to prevent and/or treat conditions associated with coagulation, including the formation of the blood clot and thrombosis. The present disclosure is also advantageous in that, in some embodiments, it utilizes the linker unit proposed in the present disclosure, which provides a facile means for controlling the amount of the cytotoxic drug payload of the present Fc-based molecular constructs. Depending on the targeting and/or effector elements selected, the present Fc-based molecular construct may take different configurations, which are discussed below, respectively.

In a first series of Fc-based molecular constructs, the targeting element is an antibody or a fragment thereof, whereas the effector element is a peptide.

Referring to FIG. 3A, which is a schematic diagram illustrating an Fc-based molecular construct 1200A comprises a pair of targeting elements T1 (as scFvs) linked to the N-termini of the pair of CH2-CH3 segments 1210, and a pair of effector elements E1 (in the form of therapeutic peptides) linked to the C-termini of the pair of CH2-CH3 segments 1210. Alternatively, in the Fc-based molecular construct 1200B of FIG. 3B, the pair of targeting elements T1 (as scFvs) is linked to the C-termini of the pair of CH2-CH3 segments 1210, whereas the pair of effector elements E1 (in the form of therapeutic peptides) is linked to the C-termini of the pair of CH2-CH3 segments 1210.

In some embodiments, the CH2-CH3 chains are adopted from human immunoglobulins γ1 or γ4. In general, γ1 is chosen, when Fc-mediated functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated activity (inflammatory activation or target cell lysis), are desired. In the case where Fc-mediated functions are avoided, γ4 is chosen for constructing the present Fc-based molecular constructs.

In some embodiments, the pair of the targeting elements takes a Fab configuration (i.e., consisting of the V_(H)—CH1 domain and the V_(L)—Cκ domain); this Fab fragment is linked to the N-termini of the CH2-CH3 chains, so that the Fc-based molecular construct adopts an IgG configuration. In these cases, the pair of effector elements may be linked to the C-termini of the pair of CH2-CH3 segments.

For example, in the Fc-based molecular construct 1200C of FIG. 3C, each of the two targeting elements T1 comprises the V_(H)—CH1 domain 820 and the V_(L)—Cκ domain 825, thereby forming a Fab configuration 830 that is linked to the N-termini of the CH2-CH3 chains 810, so that the Fc-based molecular construct 1200C adopts the IgG configuration. In this case, the pair of effector elements E1 (a therapeutic peptide) is linked to the C-termini of the pair of CH2-CH3 chains 810.

In a second series of Fc-based molecular constructs, the targeting element can be an antibody or a fragment thereof, and the elector element can be a drug bundle.

In these cases, the Fc-based molecular constructs for treating diseased cells may have the configuration of molecular construct 1000A of FIG. 4A or molecular construct 1000B of FIG. 4B. As illustrated in FIG. 4A, the effector elements E1 (for example, drug bundles) are linked to the C-termini of the pair of CH2-CH3 segments 1010, whereas the targeting elements T1 (in this case, an scFv) are linked to the N-termini of the pair of CH2-CH3 segments 1010. According to alternative embodiments, the molecular construct 1000B (see, FIG. 4B) has a pair of targeting elements T1 that takes the form of a Fab 1030. Specifically, the Fab 1030 configuration comprises the V_(H)—CH1 domain 1020 and the V_(L)—Cκ domain 1025, and is linked to the N-termini of the pair of CH2-CH3 segments 1010, so that the Fc-based molecular construct 1000A adopts the IgG configuration. In this case, the pair of effector elements E1 is linked to the C-termini of the pair of CH2-CH3 chains 1010.

In some embodiments, the CH2-CH3 chains are adopted from human immunoglobulins γ1 or γ4. In general, γ1 is chosen, when Fc-mediated functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated activity (inflammatory activation or target cell lysis), are desired. In the case where Fc-mediated functions are avoided, γ4 is chosen for constructing the present Fc-based molecular constructs.

As could be appreciated, the drug bundle (i.e., effector element E1) may be provided as the linker unit discussed in the present disclosure (see, for example FIG. 1A to FIG. 1 C). According to the principles and spirits of the present disclosure, a targeting construct (comprising the pair of CH2-CH3 segments 1010 and the targeting elements T1) and the drug bundles (for use as effector elements E1) can be prepared separately and then conjugated with each other.

According to embodiments of the present disclosure, the drug bundle comprises a center core, a plurality of linking arms, and optionally, a coupling arm. The center core may be a compound having a plurality of amine groups or a polypeptide comprising a plurality of lysine (K) residues, according to various embodiments of the present disclosure. Each of the linking arms has one terminus that is linked to the center core by reacting with the amine groups of the compound core or the amine side chain of the K residues of the polypeptide core. The linking arm also carries a maleimide group at the free terminus thereof, wherein each of the drug molecules is linked to the center core via connecting through the linking arm by reacting with the maleimide group. According to optional embodiments of the present disclosure, each of the effector elements E1 is a drug bundle with 3-5 cytotoxic molecules.

In the case where the center core is the polypeptide core, then the amino acid residue at the N- or C-terminus of the center core is a cysteine residue or has an azide group or an alkyne group. According to certain embodiments, for polypeptide cores with a terminal amino acid residue having the azide group, the drug bundle is linked to the peptide extension via the SPAAC reaction or CuAAC reaction occurred between said terminal residue and the C-terminus of the peptide extension. Alternatively, when the polypeptide cores has a terminal amino acid residue with the alkyne group, the drug bundle is linked to the peptide extension via the CuAAC reaction occurred between said terminal residue and the C-terminus of the peptide extension. Still alternatively, for polypeptide cores with a terminal residue that is cysteine or for compound cores, the drug bundle further comprises said coupling arm. Specifically, the coupling arm has one terminus linked to the center core by reacting with the cysteine residue of the polypeptide core or one amine group of the compound core. The coupling arm also carries an alkyne group, azide group, tetrazine group, or strained alkyne group at the free terminus thereof, so that the drug bundle is linked to the C-terminus of the peptide extension via the iEDDA reaction (for coupling arms with the tetrazine or cyclooctene group), SPAAC (for coupling arms with the azide or cyclooctyne group) reaction or CuAAC reaction (for coupling arms with the alkyne or azide group) occurred therebetween.

According to certain embodiments, the present Fc-based molecular construct for treating diseased cells further comprises a pair of peptide extensions 1050 (see, FIGS. 4A and 9B) respectively having the sequence of (G₂₋₄S)₂₋₈C. As illustrated, the pair of peptide extensions 1050 is linked to the C-termini of the pair of CH2-CH3 segments 1010. The cysteine residue at the C-terminus of the peptide extension is linked with a coupling arm 1055 via thiol-maleimide reaction occurred therebetween. Also, before being conjugated with the effector element E1 (in this case, a drug bundle), the free terminus of the conjugating arm (that is, the terminus that is not linked to the cysteine residue) is modified with an alkyne, azide, strained alkyne, or tetrazine group, so that the drug bundle is linked thereto via iEDDA reaction (see, FIG. 4A), SPAAC (see, FIG. 4B), or CUAAC (not shown) reaction occurred therebetween.

For example, in FIG. 4A, the coupling arm 1040 of the effector element E1 (in this case, a drug bundle) is linked to the CH2-CH3 segment 1010 via iEDDA reaction. The ellipse 1045 as depicted in FIG. 4A represents the chemical bond resulted from the iEDDA reaction occurred between the peptide extension 1050 and the effector element E1. As could be appreciated, an iEDDA reaction is occurred between a tetrazine group and a cyclooctene group, such as a transcyclooctene (TCO) group.

Alternatively, in FIG. 4B, the effector element E1 is linked to the CH2-CH3 segment 1010 via SPAAC reaction. The diamond 1045 as depicted in FIG. 4B represents the chemical bond resulted from the SPAAC reaction occurred between the peptide extension 1050 and the effector element E1. Specifically, an SPAAC reaction is occurred between an azide group and a strained alkyne group (e.g., a cyclooctyne group, including, dibenzocyclooctyne (DBCO), difluorinated cyclooctyne (DIFO), bicyclononyne (BCN), and dibenzocyclooctyne (DICO) group).

In a third series of Fc-based molecular constructs, one of the targeting and effector elements can be a peptide.

As could be appreciated, the discussions above regarding the Fc region and drug bundle of the Fc-based molecular constructs are also applicable here, and hence, detailed description regarding the same is omitted herein for the sake of brevity.

III-(ii) Functional Elements Suitable for Use with Fc-Based Molecular Construct

Now that the basic structural arrangements of the Fc-based molecular constructs have been discussed above, certain combinations of particular effector element(s) and targeting element(s) are provided below for the illustration purpose.

In constructing Fc-based molecular constructs for preventing and/or treating diseases/conditions associated with blood clots, one may use an antibody (or a fragment thereof) specific for fibrin as the targeting element.

In the case where the prevention of blood clot formation is the main purpose, the present Fc-based molecular constructs may use a drug bundle comprising multiple molecules of a Factor Xa inhibitor or thrombin inhibitor as the effector element. Illustrative examples of Factor Xa inhibitors include apixaban, edoxaban, and rivaroxaban.

Non-limiting examples of thrombin inhibitors include argatroban and melagatran. The Fc-based molecular constructs for preventing blood clot formation may take the configuration described in connection with FIG. 4A or 4B

On the other hand, for Fc-based molecular constructs aiming to treat thrombosis, the effector element can be a tissue plasminogen activator (such as alteplase, reteplase, tenecteplase and lanoteplase), which is a single-chain polypeptide. The Fc-based molecular constructs for treating thrombosis may take the configuration described in connection with FIG. 3A to 3C.

The essence of this invention is the rationalization and conception of the specific combination or pairing of the targeting and effector elements. The adoption of Fc-fusion configuration in the molecular constructs is a preferred embodiment. It is conceivable for those skilled in the arts to link the pairs of targeting and effector elements of this invention employing other molecular platforms, such as peptides, proteins (e.g., albumin), polysaccharides, polyethylene glycol, and other types of polymers, which serve as a structural base for attaching multiple molecular elements.

III-(iii) Use of Fc-Based Molecular Construct

The present disclosure also pertains to method for treating various diseases using the suitable Fc-based molecular construct. Generally, the method comprises the step of administering to a subject in need of such treatment an effective amount of the Fc-based molecular construct according to embodiments of the present disclosure.

EXPERIMENTAL EXAMPLES Example 1 Synthesis of Peptide 1 (SEQ ID NO: 18) as Peptide Core, and Conjugation of SH Group of Cysteine Residue with Maleimide-PEG₄-Tetrazine as Conjugating Arm

The synthesized peptide 1 (Chinapeptide Inc., Shanghai, China) was dissolved in 100 mM sodium phosphate buffer (pH 7.0) containing 50 mM NaCl and 5 mM EDTA at 2 mM final concentration. The dissolved peptide was reduced by 1 mM TCEP at 25° C. for 2 hours. For conjugating the SH group of cysteine residue with maleimide-PEG₄-tetrazine (Conju-probe Inc.) to create a functional linking group tetrazine, the peptide and maleimide-PEG₄-tetrazine were mixed at a 1/5 ratio and incubated at pH 7.0 and 4° C. for 24 hours. Tetrazine-conjugated peptides were purified by reverse phase HPLC on a Supelco C18 column (250 mm×10 mm; 5 μm), using a mobile phase of acetonitrile and 0.1% trifluoroacetic acid, a linear gradient of 0% to 100% acetonitrile over 30 minutes, at a flow rate of 1.0 mL/min and a column temperature of 25° C.

The present tetrazine-peptide 1, as illustrated below, had a m.w. of 2,185.2 Daltons.

Example 2 Synthesis of Peptide 2 (SEQ ID NO: 26) as Peptide Core, and Conjugation of the SH Group of Cysteine Residue with Maleimide-PEG₃-Transcyclooctene (TCO) as a Coupling Arm

The synthesized peptide 2 (Chinapeptide Inc., Shanghai, China) was processed similarly. Briefly, the peptide was dissolved in 100 mM sodium phosphate buffer (pH 7.0) containing 50 mM NaCl and 5 mM EDTA at a final concentration of 2 mM. The dissolved peptide was reduced by 1 mM tris(2-carboxyethyl)phosphine (TCEP) at 25° C. for 2 hours. For conjugating the SH group of the cysteine residue with maleimide-PEG₃-TCO (Conju-probe Inc.) to create a functional linking group TCO, the peptide and maleimide-PEG₃-TCO were mixed at a 1/7.5 ratio and incubated at pH 7.0 and 25° C. for 18 hours. TCO-conjugated peptides were purified by reverse phase HPLC on a Supelco C18 column (250 mm×10 mm; 5 μm), using a mobile phase of acetonitrile and 0.1% trifluoroacetic acid, a linear gradient of 0% to 100% acetonitrile over 30 minutes, at a flow rate of 1.0 mL/min and a column temperature of 25° C.

The identification of the synthesized TCO-peptide (illustrated below) was carried out by MALDI-TOF mass spectrometry. Mass spectrometry analyses were performed by the Mass Core Facility at the Institute of Molecular Biology (IMB), Academia Sinica, Taipei, Taiwan. Measurements were performed on a Bruker Autoflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany).

The thus-synthesized TCO-peptide 2, as illustrated below, had a m.w. of 2, 020.09 Daltons.

Example 3 Synthesis of Linker Unit by Conjugating NHS-PEG₁₂-Maleimide to NH₂ Groups of Tetrazine-Peptides 1

Three linking arms of PEG₁₂-maleimide were attached to the peptide core tetrazine-peptide 1. The crosslinker, NHS-PEG₁₂-maleimide (succinimidyl-[(N-maleimido-propionamido)-dodecaethyleneglycol] ester, was purchased from Conju-probe Inc. The conjugation procedure was performed per the manufacturer's instruction; the peptide with lysine residues was dissolved in the conjugation buffer, phosphate buffered saline (PBS, pH 7.5) at 100 mM. NHS-PEG₁₂-maleimide crosslinker was added to the dissolved peptide at 1 mM final concentration (10-fold molar excess over 0.1 mM peptide solution). The reaction mixtures were incubated for 18 hours at room temperature. PEG₁₂-maleimide-conjugated tetrazine-peptide 1 was purified by reverse phase HPLC on a Supelco C18 column (250 mm×4.6 mm; 5 μm), using a mobile phase of acetonitrile and 0.1% trifluoroacetic acid, a linear gradient of 0% to 100% acetonitrile over 30 minutes, at a flow rate of 1.0 ml/min and a column temperature of 25° C.

As illustrated below, the present PEG₁₂-maleimide-conjugated tetrazine-peptide 1 carried one coupling arm with a tetrazine group and three PEG linking arms with maleimide groups; it had a m.w. of 4,461 Daltons.

Example 4 Synthesis of Linker Unit by Conjugating NHS-PEG₆-Mal to NH₂ Groups of TCO-Peptide 2

The procedure for conjugating NHS-PEG₆-Mal to NH₂ groups of TCO-peptide 2 was performed similarly as described in the previous Example. Briefly, NHS-PEG₆-maleimide crosslinker was added to the dissolved peptide at 40 mM final concentration (20-fold molar excess over 2 mM peptide solution). The reaction mixtures were incubated for 3 hours at room temperature.

The present PEG₆-maleimide-conjugated peptide 2, as illustrated below, had a m.w. of 4,478 Daltons; it was a peptide core-based linker unit carrying one TCO group and five PEG linking arms with maleimide groups (FIG. 5).

Example 5 Conjugation of Apixaban Carboxylic Acid Molecule with NH₂-PEG₃-S—S-PEG₃-NH₂ Crosslinker

Apixaban carboxylic acid was purchased from KM3 Scientific Inc. (New Taipei City, Taiwan). The activated carboxyl group of apixaban carboxylic acid molecule was reacted with a homo-bifunctional cleavable crosslinker, NH₂-PEG₃-S—S-PEG₃-NH₂ as shown in scheme 8.

Apixaban carboxylic acid was dissolved in 100% DMSO at a final concentration of 20 mM, and NH₂-PEG₃-S—S-PEG₃-NH₂, a homo-bifunctional cleavable crosslinker, was dissolved in PBS at a 10 mM final concentration. To activate the carboxyl group of apixaban carboxylic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (KM3 Scientific Inc.) was added to the apixaban carboxylic acid solution at a molar ratio of 1:2 ([apixaban]:[EDC]) and then incubated for 15 minutes.

The activated apixaban carboxylic acid solution was added to the NH₂-PEG₃-S—S-PEG₃-NH₂ crosslinker at a 2 mM final concentration (5-fold molar excess over 0.4 mM NH₂-PEG₃-S—S-PEG₃-NH₂ crosslinker solution). The reaction mixture was incubated for 3 hours at room temperature.

Apixaban-PEG₃-S—S-PEG₃-apixaban was purified by reverse phase HPLC on a Supelco C18 column (250 mm×4.6 mm; 5 μm), using a mobile phase of acetonitrile and 0.1% trifluoroacetic acid, a linear gradient of 0% to 100% acetonitrile over 30 minutes, at a flow rate of 1.0 ml/min and a column temperature of 25° C.

The mass spectroscopic analysis of the thus-synthesized apixaban-PEG₃-S—S-PEG₃-apixaban (see, FIG. 6) indicated that the molecular construct had m.w. of 1,301.64 and 1,323.68 Daltons, corresponding to [M+H]⁺ and [M+Na]⁺, respectively.

Example 6 Conjugation of Two Argatroban Molecules to an NH₂-PEG₃-S—S-PEG₃-NH₂ Crosslinker

Argatroban was purchased from KM3 Scientific Inc. (New Taipei City, Taiwan). The procedure for conjugating NH₂-PEG₃-S—S-PEG₃-NH₂ to COOH groups of argatroban molecule was performed similarly as described in the previous Example. Briefly, argatroban was dissolved in 100% DMSO at a final concentration of 20 mM. EDC solution was added to the argatroban solution to activate COOH group of argatroban and then incubated for 15 minutes. The activated argatroban solution was added to NH₂-PEG₃-S—S-PEG₃-NH₂ crosslinker solution at a 2 mM final concentration (5-fold molar excess over 0.4 mM NH₂-PEG₃-S—S-PEG₃-NH₂ crosslinker solution) (see, scheme 9).

The MALDI-TOF result provided in FIG. 7 shows that the thus-synthesized argatroban-PEG₃-S—S-PEG₃-argatroban had a m.w. of 1,378.61 Daltons.

Example 7 Conjugation of Apixaban-PEG₃-SH and Argatroban-PEG₃-SH to Maleimide-PEG₆-Conjugated TCO-Peptide 2

Prior to conjugation with the TCO-peptide 2 that had five maleimide-PEG₆ linking arms, apixaban-PEG₃-S—S-PEG₃-apixaban and argatroban-PEG₃-S—S-PEG₃-argatroban (prepared in the preceding Examples) were incubated with 4 mM TCEP at a molar ratio of 3:1 ([TCEP]:[drug-linker]) at room temperature for 90 minutes with gentle shaking to generate the apixaban-PEG₃-SH and argatroban-PEG₃-SH molecule with a free sulfhydryl group.

The thus-synthesized drug bundle, as illustrated below, was composed of a linker unit with a free TCO functional group and a set of five apixaban molecules as effector elements. The present molecular construct had a m.w. of 7,713 Daltons, corresponding to [M+H]⁺.

Another thus-synthesized drug bundle, as illustrated below, was composed of a linker unit with a free TCO functional group and a set of five argatroban molecules as effector elements. The present molecular construct had a m.w. of 8,112.8 Daltons, corresponding to [M+H]⁺.

Example 8 Production of Mouse scFv of mAb Specific for Human Fibrin by Expi293F Overexpression System

The V_(L) and V_(H) of the scFv specific for human fibrin were from mouse monoclonal antibody 102-10 (Japanese Patent Application Publication No. 2012-72). The scFv derived from this antibody was designed to contain a flexible linker of GGGGSGGGGS and a terminal cysteine residue at the C-terminus. The cysteine residue provides a sulfhydryl group for conjugation with maleimide group present at the free ends of liking arms in various linker units. To produce the scFv of mAb specific for human fibrin, we used the V_(H) and V_(L) DNA sequences of mAb 102-10 with further codon optimization. DNA sequences encoding V_(L)-GSTSGSGKPGSGEGSTKG-V_(H)-(GGGGS)₂-C were synthesized. The amino acid sequence of the scFv of mAb 102-10 prepared for the experiments in the present invention is set forth in SEQ ID NO: 27.

For preparing scFv proteins using a mammalian expression system, we used the overexpression system based on Expi293F™ cell line for experimentation. The system employed ExpiFectamine™ 293 transfection kit (Life Technologies, Carlsbad, USA) consisting of the Expi293F™ cell line, the cationic lipid-based ExpiFectamine™ 293 Reagent and ExpiFectamine™ 293 transfection Enhancers 1 and 2, and the medium (Gibco, New York, USA).

The scFv-encoding sequence was placed in pG1K expression cassette. Expi293F cells were seeded at a density of 2.0×10⁶ viable cells/ml in Expi293F expression medium and maintained for 18 to 24 hours prior to transfection to ensure that the cells were actively dividing at the time of transfection. On the day of transfection, 7.5×10⁸ cells in 255 ml medium in a 2-liter Erlenmeyer shaker flask were transfected by ExpiFectamine™ 293 transfection reagent. The transfected cells were incubated at 37° C. for 16 to 18 hours post-transfection in an orbital shaker (125 rpm) and the cells were added ExpiFectamine™ 293 transfection enhancer 1 and enhancer 2 to the shaker flask, and incubated for another 5 to 6 days. Culture supernatants were harvested and scFv proteins in the media were purified using Protein L affinity chromatography. FIGS. 8A and 8B respectively show the results of SDS-PAGE and Mass spectrometric analysis of purified scFv of mAb specific for human fibrin. The scFv of mAb specific for human fibrin in SDS-PAGE migrated in two major bands of 26 and 30 kDa. The same protein solution was further analyzed by MALDI-TOF and showed that the 102-10 scFv specific for human fibrin had only the molecular weight of 26,838 Daltons, which is consistent with the calculated molecular weight.

Example 9 ELISA Analysis of Purified Mouse scFvs Specific for Human Fibrin

To prepare fibrinogen-coated plates, each plate was prepared according to procedures described in US patent application publication 2016/0011217A1. Briefly, 100 μl of human fibrinogen (Sigma) in PBS was added to 96-well flat-bottom plates (Nunc) at 1 μg/well, and the plate was sealed and allowed to stand at 4° C. overnight.

The fibrin plate was prepared as follows. The fibrinogen solution was removed and then 100 μL of TBS containing 0.05 U/ml thrombin (Sigma), 2 mM CaCl₂ and 7 mM L-cysteine (Sigma) was added to the wells. The thrombin-treated plate was incubated at 37° C. for 1 hour to allow fibrin formation. The thrombin solution was then removed and blocked with 10% skim milk at room temperature for 1 hour.

Then, 100 μl of the 102-10 scFv solution was added to the fibrinogen plate and the fibrin plate, which were then shaken at room temperature for 1 hour. After that, each plate was washed with TBS-T, and 50 μl of TMB (Thermo Fisher Scientific Inc., Waltham, USA) was added, and colorimetry was conducted. The reaction was stopped by adding 50 μl of 1N HCl. Then the absorbance (O.D.) was obtained by measuring the absorbance at 450 nm with a plate reader.

FIG. 8C shows the ELISA result, indicating that the purified 102-10 scFv bound specifically to human fibrin, but not to fibrinogen.

Example 10 Construction and Selection of Phage-Displayed Human scFvs Specific for Human Fibrin

The phage clones carrying the human scFv specific for human fibrin were obtained through a contractual arrangement with Dr. An-Suei Yang's laboratory at the Genomics Research Center, Academia Sinica, Taipei, Taiwan. The framework sequence of the GH2 scFv library was derived from a human IgG antibody fragment, G6 anti-VEGF Fab (Protein Bank Code 2FJG) and cloned into restriction sites SfiI and NotI of phagemid vector pCANTAB5E (GE Healthcare), carrying an ampicillin resistance, a lacZ promotor, a pelB leader sequence for secretion of scFv fragments into culture supernatants, and an E-tag applicable for detection. The V_(H) and V_(L) domains of the scFv template were diversified separately based on the oligonucleotide-directed mutagenesis procedure; the three CDRs in each of the variable domains were diversified simultaneously. The scFv library of over 10⁹ clones was used for selections on human fibrin.

The thrombin-treated fibrin plates (1 μg/100 μl per well) were prepared as described in the preceding Examples. The fibrin plates were used for panning anti-fibrin antibodies. In brief, the fibrin-coated wells were treated with blocking buffer (5% skim milk in PBST (phosphate buffered saline with 0.1% tween-20)) for 1 hour at room temperature. Recombinant phages in the blocking buffer diluted to 8×10¹¹ CFU/ml was added to the fibrin-coated wells for 1 hour with gentle shaking; CFU stands for colony-forming unit. The wells were then washed vigorously 10 times with PBST, followed by 6 times with PBS to remove nonspecific binding phages. The bound phages were eluted using 0.1 M HCl/glycine buffer at pH 2.2, and eluted fraction was neutralized immediately by 2 M Tris-base buffer at pH 9.0. E. coli strain ER2738 (OD600=˜0.6) was used for phage infection at 37° C. for 30 minutes; non-infected E. coli was eliminated by treating with ampicillin for 30 minutes. After ampicillin treatment, helper phage M13KO7 carrying kanamycin resistance was added for another 1 hour incubation. The selected phages rescued by helper phage in the E. coli culture were amplified with vigorously shaking overnight at 37° C. in the presence of kanamycin. The amplified phages were precipitated in PEG/NaCl, and then resuspended in PBS for the next selection-amplification cycle. A total of three consecutive panning rounds were performed on human fibrin by repeating this selection-amplification procedure.

Phage-infected ER2738 colonies were enumerated by serial dilution series were counted and phage titers were calculated, yielding the output titer/ml (CFU/ml) per panning round. A 1000-fold increase in phage output title from 2.5E+06 CFU/well to 4.3E+09 CFU/well was obtained after three rounds of panning. The phage output/input titer ratios from each round are shown in FIG. 9A. For each panning round, the phage output/input titer ratios are given on the y-axis. There was clear enrichment of the positive clones over the three rounds of panning. The third panning round resulted in a 500-fold on the ratios of phage output/input titer over the first round, as the binding clones became the dominant population in the library.

In a typical selection procedure, after three rounds of antigen-panning on human fibrin-coated wells in ELISA plates, approximately 80% of the bound phage particles bound to fibrin specifically in ELISA with coated fibrin.

Example 11 Single Colony ELISA Analysis of Human Phage-Displayed scFvs Specific for Human Fibrin

E. coli strain ER2738 infected with single-clonal phages each harboring a selected scFv gene in its phagemid was grown in the mid-log phase in 2YT broth (16 g/L tryptone, 10 g/l yeast extract, 5 g/l NaCl, pH 7.0) with 100 μg/ml ampicillin in deep well at 37° C. with shaking. After broth reaching an OD600 of 1.0, IPTG was added to final concentration of 1 μg/ml. The plates were incubated at 37° C. overnight with rigorously shaking. After overnight incubation at 37° C. with vigorous shaking, the plates were centrifuged at 4,000 g for 15 minutes at 4° C.

For soluble scFv binding test, ELISA was carried out. In brief, 96-well Maxisorp 96-well plate (Nunc) was coated with fibrin (1 μg/100 μl PBS per well) or a negative control antigen human fibrinogen for 18 hours with shaking at 4° C. After treated with 300 μl of blocking buffer for 1 hour, 100 μl of secreted scFv in the supernatant was mixed with 100 μl of blocking buffer and then added to the coated plate for another 1 hour. Goat anti-E-tag antibody (conjugated with HRP, 1:4000, Cat. No. AB19400, Abcam) was added to the plate for 1 hour. TMB substrate (50 μl per well) was added to the wells and the absorbance at 450 nm was measured after reactions were stopped by adding 1N HCI (50 μl per well).

A total of 960 phage clones after the 3^(rd) round of panning were subjected to the present analysis. Among them, six scFv clones that bound to fibrin with a differential of OD450 greater than 10 over fibrinogen were further characterized by DNA sequencing of their encoding scFv genes. Four different DNA sequences were identified. FIG. 9B shows the ELISA result of an scFv clone D10. The amino acid sequence of an scFV clone D10, which binds to human fibrin with an OD450 of 1.09, is shown in as SEQ ID NO: 29.

Example 12 Production of Recombinant Reteplase by Expi293F Overexpression System

The amino acid sequence of reteplase was from DrugBank. The recombinant protein was designed to contain a flexible linker of GGGGSGGGGS and a terminal cysteine residue at the C-terminus. The cysteine residue provides a sulfhydryl group for conjugation with maleimide group present at the free ends of linking arms in various linker units. The amino acid sequences of reteplase prepared for the experiments of the invention are set forth in SEQ ID NO: 28.

In this Example, the gene-encoding sequence was placed in pcDNA3 expression cassette. For preparing reteplase protein using a mammalian expression system, we used the overexpression system based on Expi293F™ cell line for experimentation as described in the above Examples.

FIGS. 10A and 10B respectively show results of SDS-PAGE and mass spectrometric analyses of purified reteplase. The recombinant reteplase in SDS-PAGE migrated in two major bands of 43 and 48 kDa. The same protein solution was further analyzed by MALDI-TOF and showed that the recombinant reteplase had only a molecular weight of 43,415 Daltons, which is consistent with the calculated molecular weight.

Example 13 Preparation of TCO-Conjugated Reteplase

For the conjugation of SH group of reteplase with Mal-PEG₃-TCO (Conju-probe, Inc.), the cysteine residue at the C-terminal end of the purified reteplase was reduced by incubating with 5 mM dithiothreitol (DTT) at room temperature for 4 hours with gentle shaking. The buffer of reduced proteins was exchanged to sodium phosphate buffer (100 mM sodium phosphate, pH7.0, 50 mM NaCl, and 5 mM EDTA) by using NAP-10 Sephadex G-25 column. After the reduction reaction and buffer exchange, conjugation was conducted overnight at room temperature in a reaction molar ratio of 10:1 ([Mal-PEG_(S)-TCO:[protein]]. The excess crosslinker was removed by a desalting column and the TCO-conjugated protein product was analyzed.

The results of mass spectroscopy MALDI-TOF analysis indicated that the sample of TCO-conjugated reteplase protein had a m.w. of 45,055 Daltons. The purity of TCO-conjugated reteplase protein was identified through Coomassie blue staining of 10% SDS-PAGE. FIG. 11 shows mass spectrometric analysis of TCO-conjugated reteplase.

Example 14 Conjugation of Three scFvs Specific for Human Fibrin to the Three Maleimide-PEG₁₂ Linking Arms Based on Tetrazine-Peptide 1

The DNA sequence encoding SEQ ID NO: 27 was synthesized and expressed as in the above Examples. Prior to conjugation with the tetrazine-peptide 1 that had three PEG₁₂-maleimide linking arms, the cysteine residue at the C-terminal end of the purified 102-10 scFv of mAb specific for human fibrin was reduced by incubating with 5 mM DTT at a molar ratio of 2:1 ([DTT]:[scFv]) at room temperature for 4 hours with gentle shaking. Subsequently, the buffer of the reduced 102-10 scFv was exchanged to maleimide-SH coupling reaction buffer (100 mM sodium phosphate, pH 7.0, 50 mM NaCl and 5 mM EDTA) by using an NAP-10 Sephadex G-25 column (GE Healthcare). After the reduction and buffer exchange, the conjugation to the tetrazine-peptide 1 having three PEG₁₂-maleimide linking arms was conducted overnight at 4° C. at a molar ratio of 1:4 ([linker]:[Protein]).

The reaction mixture was applied to a size exclusion chromatography column S75. The PEG₁₂-maleimide-conjugated tetrazine-peptide 1 conjugated with three 102-10 scFvs specific for human fibrin was separated from the free scFv, free PEG₁₂-maleimide-conjugated tetrazine-peptide 1 and the PEG₁₂-maleimide-conjugated tetrazine-peptide 1 conjugated with one and two 102-10 scFvs specific for human fibrin by size exclusion chromatography column S75. The product (i.e., the PEG₁₂-maleimide-conjugated tetrazine-peptide 1 having a free tetrazine functional group and being conjugated with a set of three 102-10 scFvs specific for human fibrin) was purified and shown in the 10% SDS-PAGE analysis shown in FIG. 12.

Example 15 Analysis of a Targeting Linker Unit Containing Three scFvs Specific for Human Fibrin Linked to the Three Maleimide-PEG₁₂ Linking Arms Based on Tetrazine-Peptide 1 by MALDI-TOF

The sample of the targeting linker unit of three 102-10 scFvs specific human fibrin linked to the three maleimide-PEG₁₂ linking arms based on tetrazine-peptide 1 was analyzed by MALDI-TOF. The median of the experimental molecular weight was consistent with the median of theoretical molecular weight of three 102-10 scFvs specific for human fibrin conjugated to tetrazine-peptide 1 with three maleimide-PEG₁₂ linking arms. According to the mass spectrometric profile in FIG. 13, the synthesized targeting linker unit had the median molecular weight of 84,974 Daltons.

Illustrated below is the synthesized targeting linker unit that was composed of a linker unit with a free tetrazine functional group and a set of three 102-10 scFvs specific for human fibrin as targeting elements.

Example 16 Preparation of Molecular Construct with Three scFvs Specific for Human Fibrin as Targeting Elements and One Reteplase Molecule as an Effector Element

In this example, the targeting linker unit of the preceding examples and a TCO-conjugated reteplase protein was coupled via a tetrazine-TCO iEDDA reaction. Specifically, the targeting linker unit had three 102-10 scFvs specific for human fibrin and one free tetrazine group.

The procedure for tetrazine-TCO ligation was performed per the manufacturer's instructions (Jena Bioscience GmbH, Jena, Germany). Briefly, 100 μl of the targeting linker unit (0.3 mg/ml) was added to the solution containing the effector element at a molar ratio of 1:1.2 ([tetrazine]:[TCO]). The reaction mixture was incubated for 1 hour at room temperature.

Illustrated below is the present joint-linker molecular construct with three 102-10 scFvs specific for human fibrin as targeting elements and with a reteplase molecule as effector elements. In 8% SDS-PAGE analysis of the reaction mixture, a band of about 180 kDa in size was observed.

Example 17 Inhibition Assay of Apixaban-PEG₃-SH Molecule

Factor Xa catalyzes the conversion of inactive prothrombin to active thrombin. Apixaban has been used as a Factor Xa inhibitor, indirectly to decrease clot formation induced by thrombin. The synthesis of the modified apixaban molecule (apixaban-PEG₃-SH) has been shown in the preceding examples. To examine the inhibitory activities of the modified apixaban molecule (apixaban-PEG₃-SH), Factor Xa inhibition assay (BioVision, Milpitas, USA) was performed. The Factor Xa inhibition assay utilizes the ability of Factor Xa to cleave a synthetic substrate thereby releasing a fluorophore, which can be detected by a fluorescence reader. In the presence of a Factor Xa inhibitor, the extent of cleavage reaction catalyzed by Factor Xa is reduced or completely abolished.

In this example, 50 μl of Factor Xa enzyme solution (provided by manufacturer) was added to the 96-well flat-bottom plate (Nunc). 10 μl of 1 μM apixaban-PEG₃-SH and apixaban carboxylic acid were added to the plate contained Factor Xa enzyme solution and incubated for 15 minutes at room temperature. Then, 40 μl of Factor Xa substrate solution (provided by manufacturer) was added to the plate and incubated at 37° C. for 30 minutes. The fluorescence intensity of fluorophores (relative fluorescence units, RFU) was obtained by measuring the emission at 450 nm under the excitation at 350 nm with fluorescence plate reader.

FIG. 14 shows the assay results of the inhibitory activity of apixaban-PEG₃-SH. In the presence of synthetic substrate, Factor Xa activity was measured in the absence of Factor Xa inhibitor (substrate only). The result indicates that the apixaban molecule conjugated with a connecting arm had a similar biological activity to inhibit action of factor Xa as the unmodified apixaban carboxylic acid. A Factor Xa inhibitor (GGACK Dihydrochloride, provided by manufacturer) is used as the control inhibitor.

Example 18 Assay of Biological Activity of Recombinant Reteplase

Reteplase is a recombinant human tissue plasminogen activator that catalyzes the conversion of plasminogen to plasmin; this process is involved in breakdown of blood clots. To investigate the biological activity of the recombinant reteplase, a chromogenic assay in 96-well flat-bottom plate was performed.

Briefly, 1 μl of 1 μM recombinant reteplase, 25 μl of 10 μM human plasminogen (Cat. No. 7549-1, Biovision) and 62.5 μl of 100 mM Tris buffer at pH 8.5 were added and incubated in the well of the plate at 37° C. for 30 minutes. Next, 1 μl of 50 mM chromogenic substrate D-Val-Leu-Lys-p-Nitroanilide dihydrochloride (Cat. No. V7127, Sigma), a synthetic plasmin substrate, was added to the well and incubated at 25° C. for 30 minutes. 31.5 μl of 10% citric acid was then added to each well to stop the reaction. The recombinant reteplase catalyzes plasminogen to form plasmin, which in turn cleaves the chromogenic substrate to release yellow colored p-Nitroanilide, which was measured at 405 nm by a plate reader.

The result shows that recombinant reteplase exhibited a protease activity with an OD405 of 1.8, whereas the positive control protein, the commercially available tPA protein (Cat. No. T0831, Sigma) had an OD405 of 1.5.

Example 19 Construction of a Gene Segment Encoding 2-Chain IgG4.Fc Fusion Protein Containing Reteplase

The reteplase-CH2-CH3 (human γ4) recombinant chain was configured by fusing reteplase to the N-terminal of CH2 domain of IgG4.Fc through a flexible hinge region. The sequence of the recombinant chain in the IgG4.Fc fusion protein molecular construct is shown as SEQ ID NO: 30.

Illustrated below is the configuration of the prepared 2-chain (reteplase)-hIgG4.Fc molecular construct.

Example 20 Expression and Purification of Recombinant 2-Chain IgG4.Fc Fusion Protein Containing Reteplase

In this Example, the gene-encoding sequence was placed in pcDNA3 expression cassette. Expi293F cells were seeded at a density of 2.0×10⁶ viable cells/ml in Expi293F expression medium and maintained for 18 to 24 hours prior to transfection to ensure that the cells were actively dividing at the time of transfection. At the time of transfection, 7.5×10⁸ cells in 255-ml medium in a 2-liter Erlenmeyer shaker flask were transfected by ExpiFectamine™ 293 transfection reagent. The transfected cells were incubated at 37° C. for 16 to 18 hours post-transfection in an orbital shaker (125 rpm) and the cells were added ExpiFectamine™ 293 transfection enhancer 1 and enhancer 2 to the shaker flask, and incubated for 7 days. Culture supernatants were harvested and recombinant 2-chain (reteplase)-hIgG4.Fc fusion protein in the media was purified using Protein A chromatography. Following buffer exchange to PBS, the concentration of (reteplase)-hIgG4.Fc protein was determined and analyzed by 8% SDS-PAGE shown in FIG. 15. The Fc-fusion molecular construct was revealed as the major band at about 74 kDa, consistent with the expected size.

Example 21 Construction of a Gene Segment Encoding 2-Chain IgG4.Fc Fusion Protein Containing Reteplase and scFv Specific for Human Fibrin

The reteplase-CH2-CH3-scFv (human γ4) recombinant chain was configured by fusing reteplase to the N-terminal of CH2 domain of IgG4.Fc through a flexible hinge region, and the 102-10 scFv specific for human fibrin was fused to the C-terminal of CH3 domain through a flexible linker, (GGGGS)₃.

The scFvs had an orientation of V_(L)-linker-V_(H). The V_(L) and V_(H) in the scFv were connected by a hydrophilic linker, GSTSGSGKPGSGEGSTKG. The sequence of the recombinant chain in the IgG4.Fc fusion protein molecular construct is shown as SEQ ID NO: 31. The expression of the constructed gene in Expi293F cells and the purification of the expressed fusion protein were performed as in preceding Examples. Characterization of the new construct was performed with SDS-PAGE. The 8% SDA-PAGE results in FIG. 16 shows that the recombinant chain of the new construct has a size of about 100 kDa, consistent with the expected size.

Illustrated below is the configuration of the prepared 2-chain (reteplase)-hIgG4.Fc-(scFv α fibrin) molecular construct.

Example 22 Construction of a Gene Segment Encoding Fusion Protein Containing Reteplase and scFv Specific for Human Fibrin

The (reteplase)-scFv recombinant chain was configured by fusing reteplase to the N-terminal of the 102-10 scFv specific for human fibrin through a flexible linker, (GGGGS)₃.

The scFv had an orientation of V_(L)-linker-V_(H). The V_(L) and V_(H) in the scFv were connected by a hydrophilic linker, GSTSGSGKPGSGEGSTKG. The sequence of the recombinant fusion protein molecular construct is shown as SEQ ID NO: 32. Characterization of the new construct was performed with SDS-PAGE. The 8% SDA-PAGE results in FIG. 17 shows that the recombinant chain of the new construct has a size of about 72 kDa, consistent with the expected size.

Illustrated below is the configuration of the prepared (reteplase)-(scFv α fibrin) molecular construct.

Example 23 Construction of a Gene Segment Encoding 2-Chain IgG4.Fc Fusion Protein Containing Tenecteplase (TNK-tPA)

The (TNK-tPA)-CH2-CH3 (human γ4) recombinant chain was configured by fusing TNK-tPA to the N-terminal of CH2 domain of IgG4.Fc through a flexible hinge region. The sequence of the recombinant chain in the IgG4.Fc fusion protein molecular construct is shown as SEQ ID NO: 33. The 8% SDA-PAGE assay results in FIG. 18 shows that the recombinant chain of the new construct has a size of about 98 kDa, consistent with the expected size.

Illustrated below is the configuration of the prepared 2-chain (TNK-tPA)-hIgG4.Fc molecular construct.

Example 24 Construction of a Gene Segment Encoding 2-Chain IgG4.Fc Fusion Protein Containing TNK-tPA and Human scFv Specific for Human Fibrin

The (TNK-tPA)-CH2-CH3-scFv (human γ4) recombinant chain was configured by fusing TNK-tPA to the N-terminal of CH2 domain of IgG4.Fc through a flexible hinge region, and the human scFv D10 specific for human fibrin was fused to the C-terminal of CH3 domain through a flexible linker, (GGGGS)₃.

The scFvs had an orientation of V_(L)-linker-V_(H). The V_(L) and V_(H) in the scFv were connected by a hydrophilic linker, GSTSGSGKPGSGEGSTKG. The sequence of the recombinant chain in the IgG4.Fc fusion protein molecular construct is shown as SEQ ID NO: 34.

To detect the recombinant 2-chain (TNK-tPA)-hIgG4.Fc-(scFv α fibrin) expressed at low level, In-gel digestion of protein isolated by gel electrophoresis and tandem mass spectrometric analysis of trypsin-digested 2-chain (TNK-tPA)-hIgG4.Fc-(scFv α fibrin) were performed for the identification of the molecular constructs. All mass spectrometry experiments were done using a Bruker Autoflex III MALI TOF/TOF mass spectrometer (Bremen, Germany) equipped with a 200 Hz Smart Bean Laser in positive ion mode with delayed extraction in the reflection mode. Data acquisition was done manually with Flex Control 3.4, and data processing was performed with Flex-Analysis 3.4 (both Bruker Dalton).

To identify the peptide by molecular mass searching of protein fragment in protein database with the Mascot search engine, the m/z values of two protein fragments in MS/MS spectrum, corresponding to 1,617.8223 and 1,053.5074 Daltons, were matched to the amino acid sequences of two TNK-tPA fragments, VYTAQNPSAQALGLGK and QYSQPQFR. The m/z value of a protein fragment in MS/MS spectrum, corresponding to 830.4496 Daltons, was matched to the amino acid sequence of a peptide fragment in the human IgG4.Fc region, GLPSSIEK. The m/z values of two protein fragments in MS/MS spectrum, corresponding to 737.3866 and 620.3044 Daltons, were matched to the amino acid sequences of two peptide fragments in the D10 scFv region, NTAYLR and MNSLR.

Illustrated below is the configuration of the prepared 2-chain (TNK-tPA)-hIgG4.Fc-(scFv α fibrin) molecular construct.

Example 25 Construction of a Gene Segment Encoding Fusion Protein Containing TNK-tPA and scFv Specific for Human Fibrin

The (TNK-tPA)-(scFv α fibrin) recombinant chain was configured by fusing TNK-tPA to the N-terminal of the human scFv D10 specific for human fibrin through a flexible linker, (GGGGS)₃.

The scFvs had an orientation of V_(L)-linker-V_(H). The V_(L) and V_(H) in the scFv were connected by a hydrophilic linker, GSTSGSGKPGSGEGSTKG. The sequence of the recombinant fusion protein molecular construct is shown as SEQ ID NO: 35. To detect the recombinant (TNK-tPA)-(scFv α fibrin) expressed at low level, In-gel digestion of protein isolated by gel electrophoresis and tandem mass spectrometric analysis of trypsin-digested (TNK-tPA)-(scFv α fibrin) were performed and confirmed for the identification of the present molecular constructs (see, the above Example).

Illustrated below is the configuration of the prepared (TNK-tPA)-(scFv α fibrin) molecular construct.

Example 26 Assay of Biological Activity of Fusion Protein Containing Reteplase and scFv Specific for Human Fibrin

Reteplase is a recombinant human tissue plasminogen activator involved in breakdown of blood clots. To investigate the biological activity of the recombinant reteplase-containing proteins fused with scFv 102-10 specific for human fibrin, a chromogenic assay in fibrin-coated plate was performed (scheme 10).

Briefly, to prepare fibrinogen plate, 100 μl of human fibrinogen (Sigma) in PBS was added 96-well flat-bottom plates (Nunc) at 10 μg/well; the plate was then sealed and allowed to stand at 4° C. overnight. The fibrin plate was prepared as follows. The fibrinogen solution was removed, and then 100 μL of TBS containing 0.05 U/ml thrombin (Sigma), 2 mM CaCl₂ and 7 mM L-cysteine (Sigma) was added to the wells. The thrombin-treated plate was incubated at 37° C. for 1 hour to allow fibrin formation. The thrombin solution was removed and blocked with 10% skim milk at room temperature for 1 hour.

Then, 100 μl of sample solution was added to the fibrinogen plate and the fibrin plate, which were then shaken at room temperature for 1 hour. After that, each plate was washed twice with PBST, followed by one PBS washing to remove nonspecific binding proteins. 25 μl of 10 μM human plasminogen (Cat. No. 7549-1, Biovision) was added and incubated in each well of the fibrinogen and fibrin plates at 37° C. for 30 minutes. 62.5 μl of 100 mM Tris buffer at pH 8.5 and 1 μl of 50 mM chromogenic substrate D-Val-Leu-Lys-p-Nitroanilide dihydrochloride (Cat. No. V7127, Sigma), a synthetic plasmin substrate, were then added to the well and incubated at 25° C. for 30 minutes. 31.5 μl of 10% citric acid was added to each well to stop the reaction. The reteplase-containing proteins catalyze plasminogen to form plasmin, which in turn cleaves the chromogenic substrate to release yellow colored p-Nitroanilide; it was measured at 405 nm by a plate reader.

FIG. 19 shows that recombinant 2-chain (reteplase)-hIgG4.Fc, 2-chain (reteplase)-hIgG4.Fc-(scFv α fibrin) and (reteplase)-(scFv α fibrin) showed protease activity as positive control proteins, recombinant reteplase and commercially available tPA protein (Cat. No. T0831, Sigma). The hIgG4.Fc and anti-fibrin 102-10 scFv were used as negative controls. The binding assay shows that both TPA and reteplase bind to fibrin. Moreover, the fusion protein of reteplase and scFv specific for fibrin, in particular the one with the G₄S linker, exhibited better binding activity.

It will be understood that the above description of embodiments is given by way of example only and that various modifications may be made by those with ordinary skill in the art. The above specification, examples, and data provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those with ordinary skill in the art could make numerous alterations to the disclosed embodiments without departing from the spirit or scope of this invention. 

What is claimed is:
 1. A molecular construct comprising, a pair of CH2-CH3 segments of an IgG.Fc; a pair of effector elements, wherein each effector element is a tissue plasminogen activator or a drug bundle comprising a plurality of molecules of an inhibitor of Factor Xa or an inhibitor of thrombin; and a pair of targeting elements, wherein each targeting element is an antibody fragment specific for fibrin, wherein, when each effector element is the tissue plasminogen activator, then the pair of effector elements is linked to the N-termini of the pair of CH2-CH3 segments, and the pair of targeting elements is linked to the N-termini of the pair of CH2-CH3 segments, or vice versa, or when each effector element is the drug bundle, then the pair of effector elements is linked to the C-termini of the pair of CH2-CH3 segments, and the pair of targeting elements is linked to the N-termini of the pair of CH2-CH3 segments.
 2. The molecular construct of claim 1, wherein the pair of CH2-CH3 segments is derived from human γ1 or γ4 immunoglobulin.
 3. The molecular construct of claim 1, wherein the pair of targeting elements is in the form of an antigen-binding fragment (Fab) and is linked to the N-termini of the pair of CH2-CH3 segments, so that the molecular construct adopts an IgG configuration.
 4. The molecular construct of claim 1, further comprising, a peptide extension, having the sequence of (G₂₋₄S)₂₋₈C, and linked to the C-terminus of one of the pair of CH2-CH3 segments; a coupling arm, linked to the C-terminus of the peptide extension via thiol-maleimide reaction occurred therebetween, wherein the drug bundle is linked to the coupling arm via inverse electron demand Diels-Alder (iEDDA) reaction, strain-promoted azide-alkyne click chemistry (SPAAC) reaction, or Copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction occurred therebetween.
 5. The molecular construct of claim 4, wherein the drug bundle comprises, a center core that is a compound having a plurality of amine groups or is a polypeptide comprising a plurality of lysine (K) residues; and a plurality of linking arms, each having one terminus linked to the center core by reacting with one of the amine groups or one of the K residues, and carrying a maleimide group at the free terminus thereof, wherein each of the molecules is linked to the center core via connecting through the linking arm by reacting with the maleimide group.
 6. The molecular construct of claim 1, wherein the inhibitor of Factor Xa is apixaban, edoxaban, or rivaroxaban.
 7. The molecular construct of claim 1, wherein the inhibitor of thrombin is argatroban or melagatran.
 8. The molecular construct of claim 1, wherein the tissue plasminogen activator is alteplase, reteplase, tenecteplase, or lanoteplase.
 9. A method for inhibiting the formation of blood clot in a subject in need thereof, comprising the step of administering to the subject an effective amount of the molecular construct according to claim 1, wherein the effector element is the drug bundle comprising the plurality of molecules of the inhibitor of Factor Xa or the inhibitor of thrombin.
 10. A method for treating thrombosis in a subject in need thereof, comprising the step of administering to the subject an effective amount of the molecular construct according to claim 1, wherein the effector is a tissue plasminogen activator. 